Cd25 pre-selective combination anti-hiv vectors, targeting vectors, and methods of use

ABSTRACT

Recombinant vectors containing at least: a backbone comprising essential sequences for integration into a target cell genome; a nucleic acid encoding a CCR5 RNAi operatively linked to a a first expression control element that regulates expression of the nucleic acid encoding the RNAi of the CCR5; a nucleic acid encoding at least the extracellular domain of CD25 operatively linked to a second expression control element that regulates expression of the nucleic acid encoding at least the extracellular domain of CD25 are provided by this disclosure. In an alternative aspect, the vector also contains polynucleotides encoding TRIM5alpha and HIV TAR decoy sequences along with gene expression regulation elements such as promoters operatively linked to the polynucleotides. The vectors are combined with packaging plasmid and envelope plasmids and optionally conjugated to cell-specific targeting antibodies. Diagnostic and therapeutic methods for using the compositions are further provided herein.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Application Ser. No. 61/681,586, filed Aug. 9, 2012, thecontent of which is incorporated by reference into the presentapplication in its entirety.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under Grant No. TR000002 awarded by the National Center for Advancing TranslationalSciences, National Institutes of Health. The government has certainrights in this invention.

BACKGROUND

Throughout this disclosure, various publications, patents and publishedpatent specifications are referenced by an identifying citation and insome aspects, the complete bibliographic of the citation is found in thesection immediately preceding the claims. The disclosures of thesepublications, patents and published patent specifications are herebyincorporated by reference into the present disclosure to more fullydescribe the state of the art to which this invention pertains.

HIV infections continue to spread worldwide in both developed andunderdeveloped countries with no effective vaccine available (Barouch etal., 2008; Edgeworth et al., 2002). Although antiretroviral therapy(ART) is effective in the majority of HIV infected patients, challengesto therapeutic and curative success include the continuing emergence ofdrug-resistant HIV variants, drug toxicity, and incomplete viralsuppression (Baldanti et al., 2010; Domingo et al., 2012; Johnson etal., 2010; Kuritzkes, 2011; Lewden et al., 2007; Macias et al., 2006;Tilton et al., 2010). ART also fails to eradicate viral reservoirs whichare established early in infection, leading to viral persistence andincomplete immune restoration (Gazzola et al., 2009; Mehandru et al.,2006). Interruption of ART results in rapid viral resurgence, thegeneration of escape mutants, and CD4+ T cell loss in the peripheralblood of HIV infected patients (Graham et al., 2012; Kalmar et al.,2012).

These challenges highlight the need for the further development ofinnovative HIV therapies with broad mechanisms of action. This inventionsatisfies this need and provides related advantages as well.

SUMMARY

HIV-1 gene therapy offers a promising alternative to small moleculeantiretroviral treatments and current vaccination strategies bytransferring, into HIV-1 susceptible cells, the genetic ability toresist infection. The need for novel and innovative strategies toprevent and treat HIV-1 infection is critical due to devastating effectsof the virus in developing countries, high cost, toxicity, andgeneration of escape mutants from antiretroviral therapies and thefailure of past and current vaccination efforts. Described herein areDNA vectors, viral packaging systems, and methods useful for HIV stemcell gene therapy with an enriched population of HIV-resistant cellscompared to unpurified cells. This was achieved by a triple combinationanti-HIV vector which incorporates a selectable marker, e.g., humanCD25, which is expressed on the surface of transduced cells. Human CD25,the low affinity IL-2 receptor alpha subunit, is an example of aselectable marker and is useful because of its normal characteristics ofnot being expressed on the surface of HPCs or HSCs and its lack ofintracellular signaling (Grant et al., 1992; Kuziel et al., 1990; Minamiet al., 1993). Upon expressing CD25 on the surface of HPCs andpurification of the transduced cells, safety of the enriched populationof anti-HIV vector transduced HPCs was observed along with potent HIV-1inhibition. This demonstrates the use of this strategy for HIV stem cellgene therapy to improve the efficacy of future HIV stem cell genetherapy clinical trials.

Thus, this invention provides a vector comprising, or alternativelyconsisting essentially of, or yet further consisting of: a vectorbackbone comprising or alternatively consisting essentially of, or yetfurther consisting of essential sequences for integration of exongenousgenes into a target cell's genome; a nucleic acid encoding a CCR5 RNAithat, in one aspect, inhibits integration of a human immunodeficiencyvirus (HIV) into a mammalian cell; a first expression control elementthat regulates expression of the nucleic acid encoding the CCR5 RNAielement and operatively linked to the CCR5 RNAi element; a nucleic acidencoding at least the extracellular domain of CD25 or an equivalentthereof; and a second expression control element that regulatesexpression of the nucleic acid encoding at least the extracellulardomain of CD25 or an equivalent thereof and operatively linked to it. Ina further aspect, the vector also contains a nucleic acid encoding aTRIM5alpha sequence and an HIV TAR sequence.

The invention also provides a vector as described above, wherein thebackbone is derived from a virus; and a packaging plasmid which containsthe nucleoside, capsid and matrix proteins. In a further aspect, theinvention further comprise an envelope plasmid and in a further aspect,a packaging cell line is provided.

Also provided are pseudotyped viral particles that are optionallyconjugated to cell-specific targeting antibodies. The particlesoptionally can be conjugated to cells. This invention also provides acell or an enriched population of HIV-resistant cells, wherein thecell(s) expresses CD25 extracellular domain on the surface of the celland comprise an HIV CCR5 RNAi element. In one aspect, the cell(s)further comprises a TRIM5alpha and/or an HIV TAR polynucleotide(s). Inone aspect the cell is a stem cell such as a hematopoietc progenitorcell (HPC) or a hematopoietic stem cell (HSC).

The vectors, particles and cells are useful to inhibit, ex vivo and invivo, the replication of HIV in a cell system, such as a cell culture,or in a subject in need thereof by administering an effective amount ofthe vector, the particle, the cell, the enriched population of cells, orthe cell conjugated to the particle. In one aspect, the methods not onlyinhibit HIV replication but also prevent replication in a subjectinfected with the virus. Specific details of the various embodiments ofthis invention are provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C depict pre-selective lentiviral vectors and purification oftransduced HPCs. FIG. 1A: A self-inactivating third generationlentiviral vector, CCLc-MNDU3-X-PGK-X2, was utilized to derive thepre-selective vectors. The control EGFP+ vector contains an EGFPreporter gene under the control of the MNDU3 promoter and a human CD25gene under the control of the PGK promoter. The CMAP1 anti-HIV vectorcontains a triple combination of anti-HIV genes, a human/rhesus macaquechimeric TRIM5α under the control of the MNDU3 promoter, a pol-III U6promoter driven CCR5 shRNA, a pol-III U6 promoter driven TAR decoy, anda human CD25 gene under the control of the PGK promoter. FIG. 1B: CD34+HPCs were transduced with the control EGFP+ pre-selective vector,purified by CD25 immunomagnetic beads, and analyzed by flow cytometryfor EGFP and CD25 expression. FIG. 1C: CD34+ HPCs were transduced withthe anti-HIV CMAP1 vector, purified by CD25 immunomagnetic beads, andanalyzed by QPCR for vector copy number. All experiments were performedin triplicate.

FIG. 2A-2C show the safety analysis of purified CMAP1 transduced HPCs.FIG. 2A: HPCs, either nontransduced (NT) or purified CMAP1 vectortransduced, were cultured in semi-solid methylcellulose medium for 10days and CFUs (blood forming colonies (BFU),granulocyte/erythrocyte/megakaryocyte/monocyte colonies (GEMM), andgranulocyte/monocyte colonies (GM)) were visualized by microscopy under10× magnification. FIG. 2B: Total BFU, GM, and GEMM colonies werecounted on day 10 for the NT and purified CMAP1 cultures. FIG. 2C: Totalcells were counted in HPC methylcellulose NT and purified CMAP1 vectortransduced cultures in the absence or presence of IL-2. All experimentswere performed in triplicate. Representative cell pictures aredisplayed.

FIGS. 3A and B depict the derivation of phenotypically normalmacrophages. FIG. 3A: Macrophages derived from the nontransduced (NT)and purified CMAP1 HPC cultures were visualized by microscopy under 10×magnification. FIG. 3B: The NT (unshaded) and purified CMAP1 (shaded)macrophages were analyzed by flow cytometry for the cell surface markersCD14, HLADR, CD4, CD80, and CCR5. FIG. 3C: Macrophages, NT, unpurifiedCMAP1 transduced, and purified CMAP1 transduced, were also analyzed byflow cytometry for the expression of CD25. All experiments wereperformed in triplicate. Representative cell pictures and flow cytometryhistogram overlays are displayed.

FIG. 4 shows the expression of proto-oncogenes in purified CMAP1macrophages. Cell cultures containing either peripheral bloodmononuclear cells (PBMCs), nontransduced (NT) HPC derived macrophages,or purified CMAP1 vector transduced HPC derived macrophages wereevaluated by QPCR for their expression of the proto-oncogenes myc, myb,fos, and jun in the presence of IL-2. Experiments were performed intriplicate. Statistical significance (p<0.05) is represented by anasterisk.

FIGS. 5A and B depict HIV-1 challenge of CMAP1 HPC derived macrophages.FIG. 5A: HPC derived macrophages, either nontransduced (NT) (♦),unpurified CMAP1 transduced (CMAP1-UP) (▪), or purified CMAP1 (CMAP1-P)(▴) transduced, were challenged with an R5-tropic BaL-1 strain of HIV-1at an MOI of 0.05. On various days post-infection, culture supernatantswere analyzed for HIV-1 replication by p24 ELISA. FIG. 5B: On day 28post-infection infected cultures were visualized by microscopy under 10×magnification. All experiments were performed in triplicate.Representative cell pictures are displayed.

FIG. 6 shows the nucleotide sequence of the full CD25 pre-selectiveanti-HIV lentiviral vector backbone (9897 base pairs). The sequence isannotated as follows: 1) the underline portion is human/rhesus macaqueTRIM5alpha; 2) the italic portion is the 2A protease sequence; 3) thedouble underline protein is full CD25 sequence; 4) the blackbackground/white type portion is the CCR5 shRNA; and 5) the shadedportion is TAR decoy sequence. This nucleotide sequence represents SEQID NO: 1.

FIG. 7 shows the nucleotide sequence of the partial truncated CD25pre-selective anti-HIV lentiviral vector backbone (9870 base pairs). Thesequence is annotated as follows: 1) the underline portion ishuman/rhesus macaque TRIM5alpha sequence; 2) the italic portion is the2A protease sequence; 3) the double underline portion is partialtruncated CD25 sequence; 4) the black background/white type portion isthe CCR5 shRNA; and 5) the shaded portion is TAR decoy sequence. Thisnucleotide sequence represends SEQ ID NO: 2.

FIG. 8 shows the nucleotide sequence of the truncated CD25 pre-selectiveanti-HIV lentiviral vector backbone (9858 base pairs). The sequence isannotated as follows: 1) the underline portion is human/rhesus macaqueTRIM5alpha sequence; 2) the italic portion is the 2A protease sequence;3) the double underline portion is truncated CD25 sequence; 4) the blackbackground/white type portion is the CCR5 shRNA sequence; and 5) theshaded portion is TAR decoy sequence. This nucleotide sequencerepresends SEQ ID NO: 3.

FIG. 9 depicts a vector map of a CD25-pre-selective-anti-HIV lentiviralvector that was constructed by the method of Example 11.

FIG. 10 depicts SEQ ID NO: 21 which is the sequence of the controlvector with EGFP from FIG. 1A: CCLc-MNDU3-EGFP-PGK-CD25. The MNDU3promoter is double underlined and shaded. EGFP is underlined, the PGKpromoter is shaded grey, and the full CD25 sequence is in doubleunderline. The 5′ and 3′ ends of the viral backbone are highlighted andunderlined with a broken line.

FIG. 11 depicts SEQ ID NO: 22 which is the sequence of the vector fromFIG. 1A: CC1c-MNDU3-antiHIV-PGK-fullCD25. Human/rhesus macaqueTRIM5alpha is underlined. CCR5 shRNA is in italics. TAR decoy is shadedgrey. The PGK promoter is double underlined. The full CD25 sequence isshown with white letters on a black background.

BRIEF DESCRIPTION OF SELECTED SEQUENCE LISTINGS

SEQ ID NO: 1 is the nucleotide sequence of the full CD25 pre-selectiveanti-HIV lentiviral vector backbone (9897 base pairs). The sequence isannotated as follows: 1) the underline portion is human/rhesus macaqueTRIM5alpha sequence; 2) the italic portion is the 2A protease sequence;3) the double underline portion is full CD25 sequence; 4) the blackbackground/white type portion is the CCR5 shRNA sequence; and 5) theshaded portion is TAR decoy sequence.

SEQ ID NO: 2 is the nucleotide sequence of the partial truncated CD25pre-selective anti-HIV lentiviral vector backbone (9870 base pairs). Thesequence is annotated as follows: 1) the underline portion ishuman/rhesus macaque TRIM5alpha sequence; 2) the italic portion is the2Aprotease sequence; 3) the double underline portion is partial truncatedCD25 sequence; 4) the black background/white type portion is the CCR5shRNA; and 5) the shaded portion is TAR decoy sequence.

SEQ ID NO: 3 is the nucleotide sequence of the truncated CD25pre-selective anti-HIV lentiviral vector backbone (9858 base pairs). Thesequence is annotated as follows: 1) the underline portion ishuman/rhesus macaque TRIM5alpha sequence; 2) the italic portion is the2A protease sequence; 3) the double underline portion is the truncatedCD25 sequence; 4) the black background/white type portion is the CCR5shRNA; and 5) the shaded portion is TAR decoy sequence.

SEQ ID NO: 4 is the nucleotide sequence of the full length CD25.

SEQ ID NO: 5 is the nucleotide sequence of the partial truncated CD25.

SEQ ID NO: 6 is the nucleotide sequence of the truncated CD25.

Alternative CCR5 RNAi for use in this invention are shown in SEQ ID NOS:7-10, as well as a full length coding sequence for Human G-ProteinChemokine Receptor (CCR5), SEQ ID NO: 11 that is reproduced from GenBankAccession No. DM068065, last accessed on Apr. 29, 2009.

SEQ ID NOS: 12-14 are additional TAR sequences for use in thisinvention.

SEQ ID NO: 15 is the sequence of a packaging plasmid sequence for use inthis invention.

SEQ ID NO: 16 is an embodiment of polynucleotides encoding thepSINDBIS-ZZ envelope plasmid and transcribed by the polymerase-II CMVpromoter into one messenger RNA. The pSINDBIS-ZZ plasmid comprises theE3 gene (SEQ ID NO: 17), the E2 gene (SEQ ID NO: 18) (the ZZ domain isbetween nucleotides 220 and 597), the 6K gene (SEQ ID NO: 19) and the E1gene (SEQ ID NO: 20).

SEQ ID NO: 21 shows the sequence of the control vector with EGFP fromFIG. 1A: CCLc-MNDU3-EGFP-PGK-CD25, also shown in FIG. 10. In FIG. 10,the MNDU3 promoter is double underlined and shaded. EGFP is underlined,the PGK promoter is shaded grey, and the full CD25 sequence is in doubleunderline. The 5′ and 3′ ends of the viral backbone are highlighted andunderlined with a broken line.

SEQ ID NO: 22 shows the sequence of the vector from FIG. 1A:CC1c-MNDU3-antiHIV-PGK-fullCD25. Human/rhesus macaque TRIM5alpha isunderlined. CCR5 shRNA is in italics. TAR decoy is shaded grey. The PGKpromoter is double underlined. The full length CD25 is shown with whiteletters on a black background.

SEQ ID NO: 23 is an example of a CCR5 shRNA.

SEQ ID NO: 24 is an example of a human TRIM5alpha 11-aa patch. SEQ IDNO: 25 shows an example of a rhesus macaque TRIM5alpha 13-aa patch.

SEQ ID NO: 26 is a polynucleotide encoding an example of a human/rhesusmacaque chimeric TRIM5alpha sequence. The first six nucleotides are theKozak sequence followed by the ATG start codon. The nucleotidescorrespond to the rhesus macaque 13 amino acids inserted into the humanTRIM5alpha sequence to make the chimeric protein (994-1032). The last 39nucleotides at the end of the sequence (1492-1530) correspond to ahemmaglutinin tag which was put on the end of the protein codingsequence for detection of expression. These 39 nucleotides are followedby the TGA stop codon.

SEQ ID NO: 27 is an HIV TAR decoy sequence for use in the embodiments ofthis invention. The polymeraseIII U6 promoter is shown as nucleotides1-283 followed by the TAR decoy sequence (284-415). The TAR decoysequence is followed by a string of 6 thymidines which is the“transcriptional stop signal” for the U6 promoter.

SEQ ID NOS: 28-29 show examples of quantitative PCR (QPCR) primerssequences for the TRIM5α gene.

SEQ ID NOS: 30-31 show examples of quantitative PCR (QPCR) primerssequences for the myc gene.

SEQ ID NOS: 32-33 show examples of quantitative PCR (QPCR) primerssequences for the myb gene.

SEQ ID NOS: 34-35 show examples of quantitative PCR (QPCR) primerssequences for the fos gene.

SEQ ID NOS: 36-37 show examples of quantitative PCR (QPCR) primerssequences for the jun gene.

SEQ ID NO: 38 shows the nucleotide sequence of an equivalent truncatedhuman CD25 nucleic acid. The nucleotide sequence has 80% or moresequence identity to SEQ ID NO: 6 or alternatively, it hybridizes underconditions of high stringency to the complement of SEQ ID NO: 6.Mutations in the sequence as compared to SEQ ID NO: 6 are underlined andin lowercase.

MODES FOR CARRYING OUT THE INVENTION Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methods,devices, and materials are now described. All technical and patentpublications cited herein are incorporated herein by reference in theirentirety. Nothing herein is to be construed as an admission that theinvention is not entitled to antedate such disclosure by virtue of priorinvention.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of tissue culture, immunology,molecular biology, microbiology, cell biology and recombinant DNA, whichare within the skill of the art. See, e.g., Sambrook and Russell eds.(2001) Molecular Cloning: A Laboratory Manual, 3^(rd) edition; theseries Ausubel et al. eds. (2007) Current Protocols in MolecularBiology; the series Methods in Enzymology (Academic Press, Inc., N.Y.);MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press atOxford University Press); MacPherson et al. (1995) PCR 2: A PracticalApproach; Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual;Freshney (2005) Culture of Animal Cells: A Manual of Basic Techique,5^(th) edition; Gait ed. (1984) Oligonucleotide Synthesis; U.S. Pat. No.4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization;Anderson (1999) Nucleic Acid Hybridization; Hames and Higgins eds.(1984) Transcription and Translation; Immobilized Cells and Enzymes (IRLPress (1986)); Perbal (1984) A Practical Guide to Molecular Cloning;Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells(Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer andExpression in Mammalian Cells; Mayer and Walker eds. (1987)Immunochemical Methods in Cell and Molecular Biology (Academic Press,London); Herzenberg et al. eds (1996) Weir's Handbook of ExperimentalImmunology; Manipulating the Mouse Embryo: A Laboratory Manual, 3^(rd)edition (Cold Spring Harbor Laboratory Press (2002)); Sohail (ed.)(2004) Gene Silencing by RNA Interference: Technology and Application(CRC Press).

All numerical designations, e.g., pH, temperature, time, concentration,and molecular weight, including ranges, are approximations which arevaried (+) or (−) by increments of 0.1 or 1.0, where appropriate. It isto be understood, although not always explicitly stated that allnumerical designations are preceded by the term “about.” It also is tobe understood, although not always explicitly stated, that the reagentsdescribed herein are merely exemplary and that equivalents of such areknown in the art.

As used in the specification and claims, the singular form “a”, “an” and“the” include plural references unless the context clearly dictatesotherwise. For example, the term “a cell” includes a plurality of cells,including mixtures thereof.

As used herein, the term “comprising” or “comprises” is intended to meanthat the compositions and methods include the recited elements, but notexcluding others. “Consisting essentially of” when used to definecompositions and methods, shall mean excluding other elements of anyessential significance to the combination for the stated purpose. Thus,a composition consisting essentially of the elements as defined hereinwould not exclude trace contaminants from the isolation and purificationmethod and pharmaceutically acceptable carriers, such as phosphatebuffered saline, preservatives and the like. “Consisting of” shall meanexcluding more than trace elements of other ingredients and substantialmethod steps for administering the compositions of this invention orprocess steps to produce a composition or achieve an intended result.Embodiments defined by each of these transition terms are within thescope of this invention.

The term “isolated” as used herein with respect to nucleic acids, suchas DNA or RNA, refers to molecules separated from other DNAs or RNAs,respectively that are present in the natural source of themacromolecule. The term “isolated nucleic acid” is meant to includenucleic acid fragments which are not naturally occurring as fragmentsand would not be found in the natural state. The term “isolated” is alsoused herein to refer to polypeptides, proteins and/or host cells thatare isolated from other cellular proteins and is meant to encompass bothpurified and recombinant polypeptides. In other embodiments, the term“isolated” means separated from constituents, cellular and otherwise, inwhich the cell, tissue, polynucleotide, peptide, polypeptide, protein,antibody or fragment(s) thereof, which are normally associated innature. For example, an isolated cell is a cell that is separated formtissue or cells of dissimilar phenotype or genotype. As is apparent tothose of skill in the art, a non-naturally occurring polynucleotide,peptide, polypeptide, protein, antibody or fragment(s) thereof, does notrequire “isolation” to distinguish it from its naturally occurringcounterpart.

As is known to those of skill in the art, there are 6 classes ofviruses. The DNA viruses constitute classes I and II. The RNA virusesand retroviruses make up the remaining classes. Class III viruses have adouble-stranded RNA genome. Class IV viruses have a positivesingle-stranded RNA genome, the genome itself acting as mRNA Class Vviruses have a negative single-stranded RNA genome used as a templatefor mRNA synthesis. Class VI viruses have a positive single-stranded RNAgenome but with a DNA intermediate not only in replication but also inmRNA synthesis. Retroviruses carry their genetic information in the formof RNA; however, once the virus infects a cell, the RNA isreverse-transcribed into the DNA form which integrates into the genomicDNA of the infected cell. The integrated DNA form is called a provirus.

The terms “polynucleotide”, “nucleic acid” and “oligonucleotide” areused interchangeably and refer to a polymeric form of nucleotides of anylength, either deoxyribonucleotides or ribonucleotides or analogsthereof. Polynucleotides can have any three-dimensional structure andmay perform any function, known or unknown. The following arenon-limiting examples of polynucleotides: a gene or gene fragment (forexample, a probe, primer, EST or SAGE tag), exons, introns, messengerRNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinantpolynucleotides, branched polynucleotides, plasmids, vectors, isolatedDNA of any sequence, isolated RNA of any sequence, nucleic acid probesand primers. A polynucleotide can comprise modified nucleotides, such asmethylated nucleotides and nucleotide analogs. If present, modificationsto the nucleotide structure can be imparted before or after assembly ofthe polynucleotide. The sequence of nucleotides can be interrupted bynon-nucleotide components. A polynucleotide can be further modifiedafter polymerization, such as by conjugation with a labeling component.The term also refers to both double- and single-stranded molecules.Unless otherwise specified or required, any embodiment of this inventionthat is a polynucleotide encompasses both the double-stranded form andeach of two complementary single-stranded forms known or predicted tomake up the double-stranded form.

A polynucleotide is composed of a specific sequence of four nucleotidebases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil(U) for thymine when the polynucleotide is RNA. Thus, the term“polynucleotide sequence” is the alphabetical representation of apolynucleotide molecule. This alphabetical representation can be inputinto databases in a computer having a central processing unit and usedfor bioinformatics applications such as functional genomics and homologysearching.

“Homology” or “identity” or “similarity” refers to sequence similaritybetween two peptides or between two nucleic acid molecules. Homology canbe determined by comparing a position in each sequence which may bealigned for purposes of comparison. When a position in the comparedsequence is occupied by the same base or amino acid, then the moleculesare homologous at that position. A degree of homology between sequencesis a function of the number of matching or homologous positions sharedby the sequences. An “unrelated” or “non-homologous” sequence sharesless than 40% identity, or alternatively less than 25% identity, withone of the sequences of the present invention.

A polynucleotide or polynucleotide region (or a polypeptide orpolypeptide region) has a certain percentage (for example, 70%, 75%,80%, 85%, 90%, 95%, 98% or 99%) of “sequence identity” to anothersequence means that, when aligned, that percentage of bases (or aminoacids) are the same in comparing the two sequences. This alignment andthe percent homology or sequence identity can be determined usingsoftware programs known in the art, for example those described inAusubel et al. eds. (2007) Current Protocols in Molecular Biology.Preferably, default parameters are used for alignment. One alignmentprogram is BLAST, using default parameters. In particular, programs areBLASTN and BLASTP, using the following default parameters: Geneticcode=standard; filter=none; strand=both; cutoff=60; expect=10;Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE;Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDStranslations+SwissProtein+SPupdate+PIR. Details of these programs can befound at the following Internet address:http://www.ncbi.nlm.nih.gov/cgi-bin/BLAST.

An equivalent nucleic acid, polynucleotide or oligonucleotide is onehaving at least 80% sequence identity, or alternatively at least 85%sequence identity, or alternatively at least 90% sequence identity, oralternatively at least 92% sequence identity, or alternatively at least95% sequence identity, or alternatively at least 97% sequence identity,or alternatively at least 98% sequence identity to the reference nucleicacid, polynucleotide, or oligonucleotide, or alternatively an equivalentnucleic acid hybridizes under conditions of high stringency to areference polynucleotide or its complement.

An equivalent polypeptide or protein is one having at least 80% sequenceidentity, or alternatively at least 85% sequence identity, oralternatively at least 90% sequence identity, or alternatively at least92% sequence identity, or alternatively at least 95% sequence identity,or alternatively at least 97% sequence identity, or alternatively atleast 98% sequence identity to the reference polypeptide or protein, oralternatively an equivalent polypeptide or protein is one encoded bynucleic acid that hybridizes under conditions of high stringency to apolynucleotide or its complement that encodes the reference polypeptideor protein.

The expression “amplification of polynucleotides” includes methods suchas PCR, ligation amplification (or ligase chain reaction, LCR) andamplification methods. These methods are known and widely practiced inthe art. See, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202 and Innis etal., 1990 (for PCR); and Wu et al. (1989) Genomics 4:560-569 (for LCR).In general, the PCR procedure describes a method of gene amplificationwhich is comprised of (i) sequence-specific hybridization of primers tospecific genes within a DNA sample (or library), (ii) subsequentamplification involving multiple rounds of annealing, elongation, anddenaturation using a DNA polymerase, and (iii) screening the PCRproducts for a band of the correct size. The primers used areoligonucleotides of sufficient length and appropriate sequence toprovide initiation of polymerization, i.e. each primer is specificallydesigned to be complementary to each strand of the genomic locus to beamplified.

Reagents and hardware for conducting PCR are commercially available.Primers useful to amplify sequences from a particular gene region arepreferably complementary to, and hybridize specifically to sequences inthe target region or its flanking regions. Nucleic acid sequencesgenerated by amplification may be sequenced directly. Alternatively theamplified sequence(s) may be cloned prior to sequence analysis. A methodfor the direct cloning and sequence analysis of enzymatically amplifiedgenomic segments is known in the art.

A “gene” refers to a polynucleotide containing at least one open readingframe (ORF) that is capable of encoding a particular polypeptide orprotein after being transcribed and translated.

The term “express” refers to the production of a gene product.

As used herein, “expression” refers to the process by whichpolynucleotides are transcribed into mRNA and/or the process by whichthe transcribed mRNA is subsequently being translated into peptides,polypeptides, or proteins. If the polynucleotide is derived from genomicDNA, expression may include splicing of the mRNA in a eukaryotic cell.

A “gene product” or alternatively a “gene expression product” refers tothe amino acid (e.g., peptide or polypeptide) generated when a gene istranscribed and translated.

“Under transcriptional control” is a term well understood in the art andindicates that transcription of a polynucleotide sequence, usually a DNAsequence, depends on its being operatively linked to an element whichcontributes to the initiation of, or promotes, transcription.“Operatively linked” intends the polynucleotides are arranged in amanner that allows them to function in a cell. In one aspect, thisinvention provides promoters operatively linked to the downstreamsequences, e.g., HIV TAR, CCR5, siRNA and TRIM5alpha.

The term “encode” as it is applied to polynucleotides refers to apolynucleotide which is said to “encode” a polypeptide if, in its nativestate or when manipulated by methods well known to those skilled in theart, it can be transcribed and/or translated to produce the mRNA for thepolypeptide and/or a fragment thereof. The antisense strand is thecomplement of such a nucleic acid, and the encoding sequence can bededuced therefrom.

A “probe” when used in the context of polynucleotide manipulation refersto an oligonucleotide that is provided as a reagent to detect a targetpotentially present in a sample of interest by hybridizing with thetarget. Usually, a probe will comprise a detectable label or a means bywhich a label can be attached, either before or subsequent to thehybridization reaction. Alternatively, a “probe” can be a biologicalcompound such as a polypeptide, antibody, or fragments thereof that iscapable of binding to the target potentially present in a sample ofinterest.

“Detectable labels” or “markers” include, but are not limited toradioisotopes, fluorochromes, chemiluminescent compounds, dyes, andproteins, including enzymes. Detectable labels can also be attached to apolynucleotide, polypeptide, antibody or composition described herein.

A “primer” is a short polynucleotide, generally with a free 3′—OH groupthat binds to a target or “template” potentially present in a sample ofinterest by hybridizing with the target, and thereafter promotingpolymerization of a polynucleotide complementary to the target. A“polymerase chain reaction” (“PCR”) is a reaction in which replicatecopies are made of a target polynucleotide using a “pair of primers” ora “set of primers” consisting of an “upstream” and a “downstream”primer, and a catalyst of polymerization, such as a DNA polymerase, andtypically a thermally-stable polymerase enzyme. Methods for PCR are wellknown in the art, and taught, for example in MacPherson et al. (1991)PCR 1: A Practical Approach (IRL Press at Oxford University Press). Allprocesses of producing replicate copies of a polynucleotide, such as PCRor gene cloning, are collectively referred to herein as “replication.” Aprimer can also be used as a probe in hybridization reactions, such asSouthern or Northern blot analyses. Sambrook and Russell (2001), infra.

“Hybridization” refers to a reaction in which one or morepolynucleotides react to form a complex that is stabilized via hydrogenbonding between the bases of the nucleotide residues. The hydrogenbonding may occur by Watson-Crick base pairing, Hoogstein binding, or inany other sequence-specific manner. The complex may comprise two strandsforming a duplex structure, three or more strands forming amulti-stranded complex, a single self-hybridizing strand, or anycombination of these. A hybridization reaction may constitute a step ina more extensive process, such as the initiation of a PCR reaction, orthe enzymatic cleavage of a polynucleotide by a ribozyme.

Hybridization reactions can be performed under conditions of different“stringency”. In general, a low stringency hybridization reaction iscarried out at about 40° C. in 10×SSC or a solution of equivalent ionicstrength/temperature. A moderate stringency hybridization is typicallyperformed at about 50° C. in 6×SSC, and a high stringency hybridizationreaction is generally performed at about 60° C. in 1×SSC. Hybridizationreactions can also be performed under “physiological conditions” whichis well known to one of skill in the art. A non-limiting example of aphysiological condition is the temperature, ionic strength, pH andconcentration of Mg²⁺ normally found in a cell.

When hybridization occurs in an antiparallel configuration between twosingle-stranded polynucleotides, the reaction is called “annealing” andthose polynucleotides are described as “complementary”. Adouble-stranded polynucleotide can be “complementary” or “homologous” toanother polynucleotide, if hybridization can occur between one of thestrands of the first polynucleotide and the second. “Complementarity” or“homology” (the degree that one polynucleotide is complementary withanother) is quantifiable in terms of the proportion of bases in opposingstrands that are expected to form hydrogen bonding with each other,according to generally accepted base-pairing rules.

The term “propagate” means to grow a cell or population of cells. Theterm “growing” also refers to the proliferation of cells in the presenceof supporting media, nutrients, growth factors, support cells, or anychemical or biological compound necessary for obtaining the desirednumber of cells or cell type.

The term “culturing” refers to the in vitro propagation of cells ororganisms on or in media of various kinds. It is understood that thedescendants of a cell grown in culture may not be completely identical(i.e., morphologically, genetically, or phenotypically) to the parentcell.

A “viral vector” is defined as a recombinantly produced virus or viralparticle that comprises a polynucleotide to be delivered into a hostcell, either in vivo, ex vivo or in vitro. Examples of viral vectorsinclude retroviral vectors, lentiviral vectors, adenovirus vectors,adeno-associated virus vectors, alphavirus vectors and the like.Alphavirus vectors, such as Semliki Forest virus-based vectors andSindbis virus-based vectors, have also been developed for use in genetherapy and immunotherapy. See, Schlesinger and Dubensky (1999) Curr.Opin. Biotechnol. 5:434-439 and Ying, et al. (1999) Nat. Med.5(7):823-827.

In aspects where gene transfer is mediated by a lentiviral vector, avector construct refers to the polynucleotide comprising the lentiviralgenome or part thereof, and a therapeutic gene. As used herein,“lentiviral mediated gene transfer” or “lentiviral transduction” carriesthe same meaning and refers to the process by which a gene or nucleicacid sequences are stably transferred into the host cell by virtue ofthe virus entering the cell and integrating its genome into the hostcell genome. The virus can enter the host cell via its normal mechanismof infection or be modified such that it binds to a different host cellsurface receptor or ligand to enter the cell. Retroviruses carry theirgenetic information in the form of RNA; however, once the virus infectsa cell, the RNA is reverse-transcribed into the DNA form whichintegrates into the genomic DNA of the infected cell. The integrated DNAform is called a provirus. As used herein, lentiviral vector refers to aviral particle capable of introducing exogenous nucleic acid into a cellthrough a viral or viral-like entry mechanism. A “lentiviral vector” isa type of retroviral vector well-known in the art that has certainadvantages in transducing nondividing cells as compared to otherretroviral vectors. See, Trono D. (2002) Lentiviral vectors, New York:Spring-Verlag Berlin Heidelberg.

Lentiviral vectors of this invention are based on or derived fromoncoretroviruses (the sub-group of retroviruses containing MLV), andlentiviruses (the sub-group of retroviruses containing HIV). Examplesinclude ASLV, SNV and RSV all of which have been split into packagingand vector components for lentiviral vector particle production systems.The lentiviral vector particle according to the invention may be basedon a genetically or otherwise (e.g. by specific choice of packaging cellsystem) altered version of a particular retrovirus.

That the vector particle according to the invention is “based on” aparticular retrovirus means that the vector is derived from thatparticular retrovirus. The genome of the vector particle comprisescomponents from that retrovirus as a backbone. The vector particlecontains essential vector components compatible with the RNA genome,including reverse transcription and integration systems. Usually thesewill include gag and pol proteins derived from the particularretrovirus. Thus, the majority of the structural components of thevector particle will normally be derived from that retrovirus, althoughthey may have been altered genetically or otherwise so as to providedesired useful properties. However, certain structural components and inparticular the env proteins, may originate from a different virus. Thevector host range and cell types infected or transduced can be alteredby using different env genes in the vector particle production system togive the vector particle a different specificity.

As used herein, “stem cell” defines a cell with the ability to dividefor indefinite periods in culture and give rise to specialized cells. Atthis time and for convenience, stem cells are categorized as somatic(adult) or embryonic. A somatic stem cell is an undifferentiated cellfound in a differentiated tissue that can renew itself (clonal) and(with certain limitations) differentiate to yield all the specializedcell types of the tissue from which it originated. An embryonic stemcell is a primitive (undifferentiated) cell from the embryo that has thepotential to become a wide variety of specialized cell types. Anembryonic stem cell is one that has been cultured under in vitroconditions that allow proliferation without differentiation for monthsto years. A clone is a line of cells that is genetically identical tothe originating cell; in this case, a stem cell.

As used herein, an “antibody” includes whole antibodies and any antigenbinding fragment or a single chain thereof. Thus the term “antibody”includes any protein or peptide containing molecule that comprises atleast a portion of an immunoglobulin molecule. Examples of such include,but are not limited to a complementarity determining region (CDR) of aheavy or light chain or a ligand binding portion thereof, a heavy chainor light chain variable region, a heavy chain or light chain constantregion, a framework (FR) region, or any portion thereof, or at least oneportion of a binding protein, any of which can be incorporated into anantibody of the present invention. The term “antibody” is furtherintended to encompass digestion fragments, specified portions,derivatives and variants thereof, including antibody mimetics orcomprising portions of antibodies that mimic the structure and/orfunction of an antibody or specified fragment or portion thereof,including single chain antibodies and fragments thereof. Examples ofbinding fragments encompassed within the term “antigen binding portion”of an antibody include a Fab fragment, a monovalent fragment consistingof the V_(L), V_(H), C_(L) and CH, domains; a F(ab′)2 fragment, abivalent fragment comprising two Fab fragments linked by a disulfidebridge at the hinge region; a Fd fragment consisting of the V_(H) andC_(H), domains; a Fv fragment consisting of the V_(L) and V_(H) domainsof a single arm of an antibody, a dAb fragment (Ward et al. (1989)Nature 341:544-546), which consists of a V_(H) domain; and an isolatedcomplementarity determining region (CDR). Furthermore, although the twodomains of the Fv fragment, V_(L) and V_(H), are coded for by separategenes, they can be joined, using recombinant methods, by a syntheticlinker that enables them to be made as a single protein chain in whichthe V_(L) and V_(H) regions pair to form monovalent molecules (known assingle chain Fv (scFv)). Bird et al. (1988) Science 242:423-426 andHuston et al. (1988) Proc. Natl. Acad Sci. USA 85:5879-5883. Singlechain antibodies are also intended to be encompassed within the term“fragment of an antibody.” Any of the above-noted antibody fragments areobtained using conventional techniques known to those of skill in theart, and the fragments are screened for binding specificity andneutralization activity in the same manner as are intact antibodies.

As used herein, an “antibody” includes whole antibodies and any antigenbinding fragment or a single chain thereof. Thus the term “antibody”includes any protein or peptide containing molecule that comprises atleast a portion of an immunoglobulin molecule. Examples of such include,but are not limited to a complementarity determining region (CDR) of aheavy or light chain or a ligand binding portion thereof, a heavy chainor light chain variable region, a heavy chain or light chain constantregion, a framework (FR) region, or any portion thereof, or at least oneportion of a binding protein, any of which can be incorporated into anantibody of the present invention. The term “antibody” is furtherintended to encompass digestion fragments, specified portions,derivatives and variants thereof, including antibody mimetics orcomprising portions of antibodies that mimic the structure and/orfunction of an antibody or specified fragment or portion thereof,including single chain antibodies and fragments thereof. It alsoincludes in some aspects, antibody variants, polyclonal antibodies,human antibodies, humanized antibodies, chimeric antibodies, antibodyderivatives, a bispecific molecule, a multispecific molecule, aheterospecific molecule, heteroantibodies and human monoclonalantibodies.

Examples of binding fragments encompassed within the term “antigenbinding portion” of an antibody include a Fab fragment, a monovalentfragment consisting of the V_(L), V_(H), C_(L) and CH, domains; aF(ab′)2 fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; a Fd fragmentconsisting of the V_(H) and C_(H), domains; a Fv fragment consisting ofthe V_(L) and V_(H) domains of a single arm of an antibody, a dAbfragment (Ward et al. (1989) Nature 341:544-546), which consists of aV_(H) domain; and an isolated complementarity determining region (CDR).Furthermore, although the two domains of the Fv fragment, V_(L) andV_(H), are coded for by separate genes, they can be joined, usingrecombinant methods, by a synthetic linker that enables them to be madeas a single protein chain in which the V_(L) and V_(H) regions pair toform monovalent molecules (known as single chain Fv (scFv)). Bird et al.(1988) Science 242:423-426 and Huston et al. (1988) Proc. Natl. AcadSci. USA 85:5879-5883. Single chain antibodies are also intended to beencompassed within the term “fragment of an antibody.” Any of theabove-noted antibody fragments are obtained using conventionaltechniques known to those of skill in the art, and the fragments arescreened for binding specificity and neutralization activity in the samemanner as are intact antibodies.

The term “antibody variant” is intended to include antibodies producedin a species other than a mouse. It also includes antibodies containingpost-translational modifications to the linear polypeptide sequence ofthe antibody or fragment. It further encompasses fully human antibodies.

The term “antibody derivative” is intended to encompass molecules thatbind an epitope as defined above and which are modifications orderivatives of a native monoclonal antibody of this invention.Derivatives include, but are not limited to, for example, bispecific,multispecific, heterospecific, trispecific, tetraspecific, multispecificantibodies, diabodies, chimeric, recombinant and humanized.

The term “bispecific molecule” is intended to include any agent, e.g., aprotein, peptide, or protein or peptide complex, which has two differentbinding specificities. The term “multispecific molecule” or“heterospecific molecule” is intended to include any agent, e.g. aprotein, peptide, or protein or peptide complex, which has more than twodifferent binding specificities.

The term “heteroantibodies” refers to two or more antibodies, antibodybinding fragments (e.g., Fab), derivatives thereof, or antigen bindingregions linked together, at least two of which have differentspecificities.

The term “human antibody” as used herein, is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences. The human antibodies of the inventionmay include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo).However, the term “human antibody” as used herein, is not intended toinclude antibodies in which CDR sequences derived from the germline ofanother mammalian species, such as a mouse, have been grafted onto humanframework sequences. Thus, as used herein, the term “human antibody”refers to an antibody in which substantially every part of the protein(e.g., CDR, framework, C_(L), C_(H) domains (e.g., C_(H1), C_(H2),C_(H3)), hinge, (VL, VH)) is substantially non-immunogenic in humans,with only minor sequence changes or variations. Similarly, antibodiesdesignated primate (monkey, baboon, chimpanzee, etc.), rodent (mouse,rat, rabbit, guinea pig, hamster, and the like) and other mammalsdesignate such species, sub-genus, genus, sub-family, family specificantibodies. Further, chimeric antibodies include any combination of theabove. Such changes or variations optionally and preferably retain orreduce the immunogenicity in humans or other species relative tonon-modified antibodies. Thus, a human antibody is distinct from achimeric or humanized antibody. It is pointed out that a human antibodycan be produced by a non-human animal or prokaryotic or eukaryotic cellthat is capable of expressing functionally rearranged humanimmunoglobulin (e.g., heavy chain and/or light chain) genes. Further,when a human antibody is a single chain antibody, it can comprise alinker peptide that is not found in native human antibodies. Forexample, an Fv can comprise a linker peptide, such as two to about eightglycine or other amino acid residues, which connects the variable regionof the heavy chain and the variable region of the light chain. Suchlinker peptides are considered to be of human origin.

As used herein, a human antibody is “derived from” a particular germlinesequence if the antibody is obtained from a system using humanimmunoglobulin sequences, e.g., by immunizing a transgenic mousecarrying human immunoglobulin genes or by screening a humanimmunoglobulin gene library. A human antibody that is “derived from” ahuman germline immunoglobulin sequence can be identified as such bycomparing the amino acid sequence of the human antibody to the aminoacid sequence of human germline immunoglobulins. A selected humanantibody typically is at least 90% identical in amino acids sequence toan amino acid sequence encoded by a human germline immunoglobulin geneand contains amino acid residues that identify the human antibody asbeing human when compared to the germline immunoglobulin amino acidsequences of other species (e.g., murine germline sequences). In certaincases, a human antibody may be at least 95%, or even at least 96%, 97%,98%, or 99% identical in amino acid sequence to the amino acid sequenceencoded by the germline immunoglobulin gene. Typically, a human antibodyderived from a particular human germline sequence will display no morethan 10 amino acid differences from the amino acid sequence encoded bythe human germline immunoglobulin gene. In certain cases, the humanantibody may display no more than 5, or even no more than 4, 3, 2, or 1amino acid difference from the amino acid sequence encoded by thegermline immunoglobulin gene.

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of singlemolecular composition. A monoclonal antibody composition displays asingle binding specificity and affinity for a particular epitope.

A “human monoclonal antibody” refers to antibodies displaying a singlebinding specificity which have variable and constant regions derivedfrom human germline immunoglobulin sequences.

The term “recombinant human antibody”, as used herein, includes allhuman antibodies that are prepared, expressed, created or isolated byrecombinant means, such as antibodies isolated from an animal (e.g., amouse) that is transgenic or transchromosomal for human immunoglobulingenes or a hybridoma prepared therefrom, antibodies isolated from a hostcell transformed to express the antibody, e.g., from a transfectoma,antibodies isolated from a recombinant, combinatorial human antibodylibrary, and antibodies prepared, expressed, created or isolated by anyother means that involve splicing of human immunoglobulin gene sequencesto other DNA sequences. Such recombinant human antibodies have variableand constant regions derived from human germline immunoglobulinsequences. In certain embodiments, however, such recombinant humanantibodies can be subjected to in vitro mutagenesis (or, when an animaltransgenic for human Ig sequences is used, in vivo somatic mutagenesis)and thus the amino acid sequences of the VH and VL regions of therecombinant antibodies are sequences that, while derived from andrelated to human germline VH and VL sequences, may not naturally existwithin the human antibody germline repertoire in vivo.

“RNA interference” (RNAi) refers to sequence-specific or gene specificsuppression of gene expression (protein synthesis) that is mediated byshort interfering RNA (siRNA).

“Short interfering RNA” (siRNA) refers to double-stranded RNA molecules(dsRNA), generally, from about 10 to about 30 nucleotides in length thatare capable of mediating RNA interference (RNAi), or 11 nucleotides inlength, 12 nucleotides in length, 13 nucleotides in length, 14nucleotides in length, 15 nucleotides in length, 16 nucleotides inlength, 17 nucleotides in length, 18 nucleotides in length, 19nucleotides in length, 20 nucleotides in length, 21 nucleotides inlength, 22 nucleotides in length, 23 nucleotides in length, 24nucleotides in length, 25 nucleotides in length, 26 nucleotides inlength, 27 nucleotides in length, 28 nucleotides in length, or 29nucleotides in length. As used herein, the term siRNA includes shorthairpin RNAs (shRNAs).

“Double stranded RNA” (dsRNA) refer to double stranded RNA moleculesthat may be of any length and may be cleaved intracellularly intosmaller RNA molecules, such as siRNA. In cells that have a competentinterferon response, longer dsRNA, such as those longer than about 30base pair in length, may trigger the interferon response. In other cellsthat do not have a competent interferon response, dsRNA may be used totrigger specific RNAi.

The term siRNA includes short hairpin RNAs (shRNAs). shRNAs comprise asingle strand of RNA that forms a stem-loop structure, where the stemconsists of the complementary sense and antisense strands that comprisea double-stranded siRNA, and the loop is a linker of varying size. Thestem structure of shRNAs generally is from about 10 to about 30nucleotides in length. For example, the stem can be 10-30 nucleotides inlength, or alternatively, 12-28 nucleotides in length, or alternatively,15-25 nucleotides in length, or alternatively, 19-23 nucleotides inlength, or alternatively, 21-23 nucleotides in length.

Tools to assist siRNA design are readily available to the public. Forexample, a computer-based siRNA design tool is available on the internetat www.dharmacon.com,Ambion-www.ambion.com/jp/techlib/misc/siRNA_finder.html; ThermoScientific-Dharmacon-www.dharmacon.com/DesignCenter/DesignCenterPage.aspx;Bioinformatics ResearchCenter-sysbio.kribb.re.kr:8080/AsiDesigner/menuDesigner.jsf; andInvitrogen-rnaidesigner.invitrogen.com/rnaiexpress/.

Without being bound by theory, it is generally believed that the primarycellular receptor for HIV entry is the CD4 receptor. In addition to CD4expression, CXCR4, and CCR5 are necessary co-factors that allow HIVentry when co-expressed with CD4 on a cell surface.

CXCR4, or fusin, is expressed on T cells (Feng et al. (1996) Science10:272(5263):872-7. Co-expression of CXCR4 and CD4 on a cell allowT-tropic HIV isolates to fuse with and infect the cell. HIV gp120interacts with both CD4 and CXCR4 to adhere to the cell and to effectconformational changes in the gp 120/gp41 complex that allow membranefusion by gp41.

CCR5 is another co-receptor that is expressed on macrophages and on somepopulations of T cells, can also function in concert with CD4 to allowHIV membrane fusion (Deng et al. (1996) Nature, June 20;381(6584):661-6.

TRIM5alpha is 493 amino acid protein that is found in most primate cellsthat appears to act to interfere with the replication of retrovirus ininfected cells. The human protein sequence is published in GenBank(Accession number NP_149023) and the mRNA sequence also has beenpublished (NM_033034). Murine protein sequence is available at NP_783608and mRNA is available at NM_175677. (All last accessed Apr. 29, 2009).

HIV is a retrovirus that is roughly spherical with a diameter of about120 nm. HIV is composed of two copies of positive single-stranded RNAthat codes for the virus' nine genes enclosed by a conical capsidcomposed of 2,000 copies of the viral protein p24. The single-strandedRNA is tightly bound to nucleocapsid proteins, p7 and enzymes needed forthe development of the virion such as reverse transcriptase, proteases,ribonuclease and integrase. A matrix composed of the viral protein p17surrounds the capsid ensuring the integrity of the virion particle. TheRNA genome of HIV consists of at least seven structural landmarks (LTR,TAR, RRE, PE, SLIP, CRS, and INS) and nine genes (gag, pol, and env,tat, rev, nef, vif, vpr, vpu, and tev) encoding 19 proteins. Three ofthese genes, gag, pol, and env, contain information needed to make thestructural proteins for new virus particles. For example, env codes fora protein called gp160 that is broken down by a viral enzyme to formgp120 and gp41. The six remaining genes, tat, rev, nef, vif, vpr, andvpu (or vpx in the case of HIV-2), are regulatory genes for proteinsthat control the ability of HIV to infect cells, produce new copies ofvirus (replicate), or cause disease. The two Tat proteins (p16 and p14)are transcriptional transactivators for the LTR promoter acting bybinding the TAR RNA element. Activation of HIV-1 gene expression by thetransactivator Tat is dependent on the RNA regulatory element (TAR)located downstream of the transcription initiation site. This elementforms a stable stem-loop structure and can be bound by either theprotein encoded by this gene or by RNA polymerase II. This protein mayact to disengage RNA polymerase II from TAR during transcriptionalelongation. Alternatively spliced transcripts of this gene may exist,but their full-length natures have not been determined. The mRNAsequence is known in the art and reported at NM_005646, last accessed onAug. 3, 2013.

An “shRNA CCR5” is an interfering RNA that down regulates or suppressesexpression of the CCR5 polynucleotide, examples of which are shown inSEQ ID NO: 11. CCR5 is also known as Human G-protein Chemokine ReceptorHDGNR10. Isolated polynucleotides encoding this protein are known in theart and described, for example in U.S. Pat. No. 7,501,123.

The term “CD25” refers to the low affinity IL-2 receptor alpha subunittransmembrane protein. CD25 is the alpha chain of the IL-2 receptor, andis not normally found on CD34+ hematopoietic progenitor cells (HPCs). Inaddition to the CD25 sequences described herein, the GenBank Accessionnumbers: NP_000408.1 and NP_032393.3 are examples of the human and mouseCD25 protein sequences, respectively. These GenBank sequences areincorporated by reference for all purposes. The extracellular domain ofCD25 refers to the first 657 nucleotides of the CD25. For example, theextracellular domain of CD25 is shown in the first 657 nucleotides ofSEQ ID NO: 4. SEQ ID NOS: 6 and 38 depict truncated versions of CD25.SEQ ID NO: 38 is an example of an equivalent of SEQ ID NO: 6.

The term “an expression control element” as used herein, intends apolynucleotide that is operatively linked to a target polynucleotide tobe transcribed, and facilitates the expression of the targetpolynucleotide. A promoter is an example of an expression controlelement.

A promoter is a regulatory polynucleotide, usually located 5′ orupstream of a gene or other polynucleotide, that provides a controlpoint for regulated gene transcription. Polymerase II and III areexamples of promoters.

A polymerase II or “pol II” promoter catalyzes the transcription of DNAto synthesize precursors of mRNA, and most shRNA and microRNA. Examplesof pol II promoters are known in the art and include without limitation,the phosphoglycerate kinase (“PGK”) promoter; EF1-alpha; CMV (minimalcytomegalovirus promoter); and LTRs from retroviral and lentiviralvectors.

A polymerase III or “pol III” promoter is a polynucleotide found ineukaryotic cells that transcribes DNA to synthesize ribosomal 5S rRNA,tRNA and other small RNAs. Examples of pol III promoters include withoutlimitation a U6 promoter or an MNDU3 promoter.

A “target cell” as used herein, shall intend a cell containing thegenome into which polynucleotides that are operatively linked to anexpression control element are to be integrated. Cells that are infectedwith HIV or susceptible to HIV infection are examples of target cells.

As used herein, the term “reporter marker” intends a polynucleotide,detectable label or other molecule that allows for the identification ofa preselected composition. Non-limiting examples of reporter markersinclude, without limitation CD25, a hemmaglutinin tag, an enhanced greenfluorescent protein (EGFP), a red flouresence protein (RFP), a greenfluorescent protein (GFP) and yellow fluorescent protein (YFP) or thelike. These are commercially available and described in the technicalart.

A “composition” is intended to mean a combination of active polypeptide,polynucleotide or antibody and another compound or composition, inert(e.g. a detectable label) or active (e.g. a gene delivery vehicle).

A “pharmaceutical composition” is intended to include the combination ofan active polypeptide, polynucleotide or antibody with a carrier, inertor active such as a solid support, making the composition suitable fordiagnostic or therapeutic use in vitro, in vivo or ex vivo.

As used herein, the term “pharmaceutically acceptable carrier”encompasses any of the standard pharmaceutical carriers, such as aphosphate buffered saline solution, water, and emulsions, such as anoil/water or water/oil emulsion, and various types of wetting agents.The compositions also can include stabilizers and preservatives. Forexamples of carriers, stabilizers and adjuvants, see Martin (1975)Remington's Pharm. Sci., 15th Ed. (Mack Publ. Co., Easton).

A “subject,” “individual” or “patient” is used interchangeably herein,and refers to a vertebrate, preferably a mammal, more preferably ahuman. Mammals include, but are not limited to, murines, rats, rabbit,simians, bovines, ovine, porcine, canines, feline, farm animals, sportanimals, pets, equine, and primate, particularly human. Besides beinguseful for human treatment, the present invention is also useful forveterinary treatment of companion mammals, exotic animals anddomesticated animals, including mammals, rodents, and the like which issusceptible to RNA and in particular, HIV viral infection. In oneembodiment, the mammals include horses, dogs, and cats. In anotherembodiment of the present invention, the human is an adolescent orinfant under the age of eighteen years of age.

“Host cell” refers not only to the particular subject cell but to theprogeny or potential progeny of such a cell. Because certainmodifications may occur in succeeding generations due to either mutationor environmental influences, such progeny may not, in fact, be identicalto the parent cell, but are still included within the scope of the termas used herein.

An “enriched population” of cells intends a substantially homogenouspopulation of cells having certain defined characteristics. The cellsare greater than 70%, or alternatively greater than 75%, oralternatively greater than 80%, or alternatively greater than 85%, oralternatively greater than 90%, or alternatively greater than 95%, oralternatively greater than 98% identical in the defined characteristics.In one aspect, the substantially homogenous population of cells expressCD25 on the surface and contain an exogenous CCR5 polynucleotide. In afurther aspect, the cells further comprise an exogenous TRIM5alphapolynucleotide and/or HIV TAR polynucleotide.

The terms “disease,” “disorder,” and “condition” are used inclusivelyand refer to any condition mediated at least in part by infection by anRNA virus such as HIV.

“Treating” or “treatment” of a disease includes: (1) preventing thedisease, i.e., causing the clinical symptoms of the disease not todevelop in a patient that may be predisposed to the disease but does notyet experience or display symptoms of the disease; (2) inhibiting thedisease, i.e., arresting or reducing the development of the disease orits clinical symptoms; or (3) relieving the disease, i.e., causingregression of the disease or its clinical symptoms.

The term “suffering” as it related to the term “treatment” refers to apatient or individual who has been diagnosed with or is predisposed toinfection or a disease incident to infection. A patient may also bereferred to being “at risk of suffering” from a disease because ofactive or latent infection. This patient has not yet developedcharacteristic disease pathology.

An “effective amount” is an amount sufficient to effect beneficial ordesired results. An effective amount can be administered in one or moreadministrations, applications or dosages. Such delivery is dependent ona number of variables including the time period for which the individualdosage unit is to be used, the bioavailability of the therapeutic agent,the route of administration, etc. It is understood, however, thatspecific dose levels of the therapeutic agents of the present inventionfor any particular subject depends upon a variety of factors includingthe activity of the specific compound employed, the age, body weight,general health, sex, and diet of the subject, the time ofadministration, the rate of excretion, the drug combination, and theseverity of the particular disorder being treated and form ofadministration. Treatment dosages generally may be titrated to optimizesafety and efficacy. Typically, dosage-effect relationships from invitro and/or in vivo tests initially can provide useful guidance on theproper doses for patient administration. In general, one will desire toadminister an amount of the compound that is effective to achieve aserum level commensurate with the concentrations found to be effectivein vitro. Determination of these parameters is well within the skill ofthe art. These considerations, as well as effective formulations andadministration procedures are well known in the art and are described instandard textbooks. Consistent with this definition, as used herein, theterm “therapeutically effective amount” is an amount sufficient toinhibit RNA virus replication ex vivo, in vitro or in vivo.

The term administration shall include without limitation, administrationby oral, parenteral (e.g., intramuscular, intraperitoneal, intravenous,ICV, intracisternal injection or infusion, subcutaneous injection, orimplant), by inhalation spray nasal, vaginal, rectal, sublingual,urethral (e.g., urethral suppository) or topical routes ofadministration (e.g., gel, ointment, cream, aerosol, etc.) and can beformulated, alone or together, in suitable dosage unit formulationscontaining conventional non-toxic pharmaceutically acceptable carriers,adjuvants, excipients, and vehicles appropriate for each route ofadministration. The invention is not limited by the route ofadministration, the formulation or dosing schedule.

Descriptive Embodiments Vectors

This invention provides a vector comprising, or alternatively consistingessentially of, or yet further consisting of a viral backbone. In oneaspect, the viral backbone contains essential nucleic acids or sequencesfor integration into a target cell's genome. In one aspect, theessential nucleic acids necessary for integration of the genome of thetarget cell include at the 5′ and 3′ ends the minimal LTR regionsrequired for integration of the vector. The vector also comprises, oralternatively consists essentially of, or yet further consists of anucleic acid encoding a CCR5 RNAi; an operatively linked firstexpression control element that regulates expression of the nucleic acidencoding the CCR5 RNAi element; a nucleic acid encoding at least theextracellular domain of CD25 or an equivalent thereof; and anoperatively linked second expression control element that regulatesexpression of the nucleic acid encoding at least the extracellulardomain of CD25 or an equivalent thereof operatively linked to it.

In one aspect, the term “vector” intends a recombinant vector thatretains the ability to infect and transduce non-dividing and/orslowly-dividing cells and integrate into the target cell's genome. Inseveral aspects, the vector is derived from or based on a wild-typevirus. In further aspects, the vector is derived from or based on awild-type lentivirus. Examples of such, include without limitation,human immunodeficiency virus (HIV), equine infectious anaemia virus(EIAV), simian immunodeficiency virus (SIV) and feline immunodeficiencyvirus (FIV). Alternatively, it is contemplated that other retrovirus canbe used as a basis for a vector backbone such murine leukemia virus(MLV). It will be evident that a viral vector according to the inventionneed not be confined to the components of a particular virus. The viralvector may comprise components derived from two or more differentviruses, and may also comprise synthetic components. Vector componentscan be manipulated to obtain desired characteristics, such as targetcell specificity.

The recombinant vectors of this invention are derived from primates andnon-primates. Examples of primate lentiviruses include the humanimmunodeficiency virus (HIV), the causative agent of human acquiredimmunodeficiency syndrome (AIDS), and the simian immunodeficiency virus(SIV). The non-primate lentiviral group includes the prototype “slowvirus” visna/maedi virus (VMV), as well as the related caprinearthritis-encephalitis virus (CAEV), equine infectious anaemia virus(EIAV) and the more recently described feline immunodeficiency virus(FIV) and bovine immunodeficiency virus (BIV). Prior art recombinantlentiviral vectors are known in the art, e.g., see U.S. Pat. Nos.6,924,123; 7,056,699; 7,07,993; 7,419,829 and 7,442,551, incorporatedherein by reference.

U.S. Pat. No. 6,924,123 discloses that certain retroviral sequencefacilitate integration into the target cell genome. This patent teachesthat each retroviral genome comprises genes called gag, pol and envwhich code for virion proteins and enzymes. These genes are flanked atboth ends by regions called long terminal repeats (LTRs). The LTRs areresponsible for proviral integration, and transcription. They also serveas enhancer-promoter sequences. In other words, the LTRs can control theexpression of the viral genes. Encapsidation of the retroviral RNAsoccurs by virtue of a psi sequence located at the 5′ end of the viralgenome. The LTRs themselves are identical sequences that can be dividedinto three elements, which are called U3, R and U5. U3 is derived fromthe sequence unique to the 3′ end of the RNA. R is derived from asequence repeated at both ends of the RNA, and U5 is derived from thesequence unique to the 5′ end of the RNA. The sizes of the threeelements can vary considerably among different retroviruses. For theviral genome. and the site of poly (A) addition (termination) is at theboundary between R and U5 in the right hand side LTR. U3 contains mostof the transcriptional control elements of the provirus, which includethe promoter and multiple enhancer sequences responsive to cellular andin some cases, viral transcriptional activator proteins.

With regard to the structural genes gag, pol and env themselves, gagencodes the internal structural protein of the virus. Gag protein isproteolytically processed into the mature proteins MA (matrix), CA(capsid) and NC (nucleocapsid). The pol gene encodes the reversetranscriptase (RT), which contains DNA polymerase, associated RNase Hand integrase (IN), which mediate replication of the genome.

For the production of viral vector particles, the vector RNA genome isexpressed from a DNA construct encoding it, in a host cell. Thecomponents of the particles not encoded by the vector genome areprovided in trans by additional nucleic acid sequences (the “packagingsystem”, which usually includes either or both of the gag/pol and envgenes) expressed in the host cell. The set of sequences required for theproduction of the viral vector particles may be introduced into the hostcell by transient transfection, or they may be integrated into the hostcell genome, or they may be provided in a mixture of ways. Thetechniques involved are known to those skilled in the art.

Retroviral vectors for use in this invention include, but are notlimited to Invitrogen's pLenti series versions 4, 6, and 6.2 “ViraPower”system. Manufactured by Lentigen Corp.; pHIV-7-GFP, lab generated andused by the City of Hope Research Institute; “Lenti-X” lentiviralvector, pLVX, manufactured by Clontech; pLKO.1-puro, manufactured bySigma-Aldrich; pLemiR, manufactured by Open Biosystems; and pLV, labgenerated and used by Charité Medical School, Institute of Virology(CBF), Berlin, Germany.

The vector also comprises, or alternatively consists essentially of, oryet further consists of, a nucleic acid encoding a CCR5 RNAi. The RNAimay be any one or more of RNA interfering molecules as described herein,e.g., shRNA, siRNA, miRNA or dsRNA. In one particular aspect, the RNAiis one or more of a shRNA or siRNA sequence shown in SEQ ID NO: 23 or anequivalent thereof, e.g., a polynucleotide having at least 80% sequenceidentity, or alternatively at least 85% sequence identity, oralternatively at least 90% sequence identity, or alternatively at least92% sequence identity, or alternatively at least 95% sequence identity,or alternatively at least 97% sequence identity, or alternatively atleast 98% sequence identity. Alternative sequences for use in thisinvention are available using the publicly available CCR5 polynucleotidesequence (see U.S. Pat. No. 7,501,123 or those disclosed in SEQ ID NOS:7-11) and a computer-based siRNA design tool available on the internetat one or more of www.dharmacon.com,Ambion-www.ambion.com/jp/techlib/misc/siRNA_finder.html; ThermoScientific-Dharmacon-www.dharmacon.com/DesignCenter/DesignCenterPage.aspx;Bioinformatics ResearchCenter-sysbio.kribb.re.kr:8080/AsiDesigner/menuDesigner.jsf; andInvitrogen-rnaidesigner.invitrogen.com/rnaiexpress/.

To produce RNAi for use in this invention, one can follow conventionaltechniques as described in the art using published sequences or thoseprovided herein. dsRNA and siRNA can be synthesized chemically orenzymatically in vitro using the methods disclosed in Micura, R. (2002)Agnes Chem. Int. Ed. Emgl. 41: 2265-9; Betz, N. (2003) Promega Notes85:15-18; Paddison, P. J. and Hannon, G. J. (2002) Cancer Cell. 2:17-23.Chemical synthesis can be performed via manual or automated methods,both of which are well known in the art. Micura, R. (2002) Agnes Chem.Int. Ed. Emgl. 41: 2265-9. siRNA can also be endogenously expressedinside the cells in the form of shRNAs. Yu, J-Y. et al. (2002) Proc.Natl. Acad. Sci. USA 99: 6047-52; McManus, M. T. et al. (2002) RNA8:842-50. Endogenous expression has been achieved using plasmid-basedexpression systems using small nuclear RNA promoters, such as RNApolymerase III U6 or H1, or RNA polymerase II U1. Brummelkamp, T. R. etal. (2002) Science 296:550-3; Novarino, G. et al. (2004) J. Neurosci.24:5322-30.

In vitro enzymatic dsRNA and siRNA synthesis can be performed using anRNA polymerase mediated process to produce individual sense andantisense strands that are annealed in vitro prior to delivery into thecells of choice. Fire A. et al. (1998) Nature 391:806-811; Donze, O. andPicard, D. (2002) Nucl. Acids Res. 30 (10):e46; Yu, J-Y. et al. (2002)Proc. Natl. Acad. Sci. USA 99: 6047-52; Shim, E. Y. et al. (2002) J.Biol. Chem. 277:30413-6. Several manufacturers (Promega Corp. (Madison,Wis.); Ambion, Inc. (Austin, Tex.); New England Biolabs (Ipswich,Mass.); and Stragene (La Jolla, Calif.) provide transcription kitsuseful in performing the in vitro synthesis.

Alternatively, one can use a Polymerase II promoter to express CCR5miRNA. For this embodiment, microRNAs are initially transcribed from PolII promoters as long transcripts called primary-miRNAs which areprocessed into small pre-miRNAs. These pre-miRNAs are further processedintracellularly into miRNAs which are the mediators of gene regulation.CCR5 shRNAs or siRNAs can be converted to miRNAs by swapping in theexact CCR5 siRNA sequence in place of the original miRNA sequence.Hence, the CCR5 siRNA can be expressed via a Pol II promoter in thecontext of a miRNA backbone for efficient processing andregulation/knockdown of gene expression. The original CCR5 siRNAsequence will be found within the entire miRNA sequence but will besurrounded by the original miRNA secondary structure generated from theextra nucleotides found in the miRNA backbone. Alternative Polymerase IIpromoters include, but are not limited to EF1-alpha; PGK(phosphoglycerate kinase promoter); CMV (minimal cytomegaloviruspromoter) and LTRs from retroviral and lentiviral vectors.

In other embodiments, the vector further comprise, or alternativelyconsists essentially of, or yet further consists of a nucleic acidencoding at least the extracellular domain of CD25 or an equivalentthereof and an operatively linked second expression control element thatregulates expression of the nucleic acid encoding at least theextracellular domain of CD25 or an equivalent thereof. In someembodiments, the first or second expression control element is apromoter. In related embodiments, the first or second expression controlelement comprises a Polymerase III promoter or a Polymerase II promoter.The Polymerase II promoter may comprise, for example, thephosphoglycerate kinase promoter (PGK). In yet further embodiments, thevector backbone is derived from a virus, such as a lentivirus. Incertain embodiments, the CD25 comprises a portion of the polynucleotideof SEQ ID NO: 1, or SEQ ID NO: 4, or SEQ ID NOS: 5, 6 or 38, or anequivalent of each thereof, e.g. a polynucleotide having at least 80%identity thereto. In other embodiments, the nucleic acid encoding CD25comprises a portion of the polynucleotide of SEQ ID NO: 3 or anequivalent thereof, e.g., a polynucleotide having at least 80% identitythereto. In a further embodiment, the nucleic acid encoding CD25comprises a portion of the polynucleotide of SEQ ID NO: 2 or anequivalent thereof, e.g. a polynucleotide having at least 80% identitythereto.

In a further embodiment, the vector further comprise, or alternativelyconsists essentially of, or yet further consists of a nucleic acidencoding a TRIM5alpha polynucleotide and a sequence the regulatesexpression of the TRIM5alpha sequence operatively linked to it. For thepurpose of illustration only, a nucleic acid that encodes TRIM5alpha foruse in this invention is one that encodes either of the amino acidsequences for human TRIM5α 11-amino acid patch (GARGTRYQTFV (SEQ ID NO:24)) or the rhesus macaque TRIM5α 13-amino acid patch (QAPGTLFTFPSLT(SEQ ID NO: 25)) or equivalents to these sequences. A TRIM5 expressioncassette having the TRIM5alpha sequence operatively linked to apolymerase-III promoter is provided in SEQ ID NOS: 1-3 and 22.Alternative nucleic acids include, but are not limited to an equivalentpolynucleotide as defined above, e.g., a polynucleotide having at least80% sequence identity, or alternatively at least 85% sequence identity,or alternatively at least 90% sequence identity, or alternatively atleast 92% sequence identity, or alternatively at least 95% sequenceidentity, or alternatively at least 97% sequence identity, oralternatively at least 98% sequence identity to the TRIM5alpha sequenceshown in SEQ ID NO: 26 as long as the 11 amino acid (human) or the 13amino acid sequence (rhesus macaque) remains.

Another embodiment of the disclosure includes vectors wherein the one ormore expression control element comprises a Polymerase II promoter thatregulates expression of a polynucleotide comprising the nucleic acidencoding the TRIM5alpha sequence and the nucleic acid encoding at leastthe extracellular domain of CD25 or an equivalent thereof. In thisinstance, the TRIM5alpha protein and the CD25 protein or an equivalentthereof is expressed as a fusion protein, and the expression iscontrolled by the same promoter. The fusion protein may furthercomprise, or alternatively consists essentially of, or yet furtherconsists of a protease cleavage site between the TRIM5alpha sequence andthe CD25 sequence or an equivalent thereof. In some embodiments, theprotease cleavage site is the 2A protease cleavage site. Other proteasecleavage sites useful in the aspects described herein include, forexample, E2A, F2A, and T2A. In other embodiments, the Polymerase IIpromoter controlling the expression of the TRIM5alpha-CD25 fusionprotein is the MNDU3 promoter. Embodiments of the vector which includethis fusion protein include, for example, the vectors of SEQ ID NOS:1-3.

In a yet further embodiment, the vector further comprise, oralternatively consists essentially of, or yet further consists of anucleic acid encoding an HIV TAR decoy polynucleotide. For the purposeof illustration only, a nucleic acid for use in this invention areprovided in a portion of SEQ ID NO: 27 or an equivalent thereof, alongwith the sequence of a suitable regulation element such as polymerase-IIpromoter operatively linked to the sequence. Alternative nucleic acidsinclude, but are not limited to a polynucleotide having at least 80%sequence identity, or alternatively at least 85% sequence identity, oralternatively at least 90% sequence identity, or alternatively at least92% sequence identity, or alternatively at least 95% sequence identity,or alternatively at least 97% sequence identity, or alternatively atleast 98% sequence identity to the TAR decoy sequence shown in SEQ IDNO: 27 or one that hybridizes under stringent conditions to SEQ ID NO:27 or its complement. Alternative sequences for use in this inventionare disclosed in U.S. Patent Publication Nos. 2004/013167 and2003/0013669 and U.S. Pat. Nos. 6,995,258; 5,994,108; and 5,693,508. HIVTAR mRNA sequences are also available on the GenBank database, availableunder Accession Nos. NM_134324 (Homo sapiens TAR (HIV-1) RNA bindingprotein 2, transcript variant 2) and NM_004178 ((Homo sapiens TAR(HIV-1) RNA binding protein 2, transcript variant 3), each last accessedon Apr. 29, 2009. These sequences are incorporated by reference intothis application. Further additional sequences include: a)5′-cgacttaaaatcgctagccagatctgagcctgggagctctctggctag-3′ (SEQ ID NO: 12)or b) 5′-gggtctctctggttagaccagatttgagcctgggagctctctggctaactagggaaccc-3′(SEQ ID NO: 13) or c) 5′-acgaagcttgatcccgtttgccggtcgatcgcttcga-3′ (SEQID NO: 14).

In a further aspect, the vector further comprises a marker or detectablelabel such as a gene encoding an enhanced green fluorescent protein(EGFP), red flouresence protein (RFP), green fluorescent protein (GFP)and yellow fluorescent protein (YFP) or the like. These are commerciallyavailable and described in the technical art.

Packaging Systems

The invention also provides a viral packaging system comprising: thevector as described above, wherein the backbone is derived from a virus;a packaging plasmid; and an envelope plasmid. The packaging plasmidcontains polynucleotides encoding the nucleoside, capsid and matrixproteins. As an example, SEQ ID NO: 15 provides the sequence encoding apackaging plasmid that can be used in this invention. Alternativesinclude, but are not limited to a polynucleotide having at least 80%sequence identity, or alternatively at least 85% sequence identity, oralternatively at least 90% sequence identity, or alternatively at least92% sequence identity, or alternatively at least 95% sequence identity,or alternatively at least 97% sequence identity, or alternatively atleast 98% sequence identity to SEQ ID NO: 15 or one that hybridizesunder stringent conditions to SEQ ID NO: 15 or its complement.Alternatives are also described in the patent literature, e.g., U.S.Pat. Nos. 7,262,049; 6,995,258; 7,252,991 and 5,710,037, incorporatedherein by reference.

The system also contains a plasmid encoding a pseudotyped envelopeprotein provided by an envelope plasmid. Pseudotyped viral vectorsconsist of vector particles bearing glycoproteins derived from otherenveloped viruses or alternatively constaining functional portions. See,for example U.S. Pat. No. 7,262,049, incorporated herein by reference.In a preferred aspect, the envelope plasmid encodes an envelope proteinthat does not cause the viral particle to unspecifically bind to a cellor population of cells. The specificity of the viral particle isconferred by the antibody binding domain that is inserted into theparticle. Examples of suitable envelope proteins include, but are notlimited to those containing the Staph. aureus ZZ domain, the encodingsequence of which is provided in SEQ ID NO: 16 or a polynucleotidehaving at least 80% sequence identity, or alternatively at least 85%sequence identity, or alternatively at least 90% sequence identity, oralternatively at least 92% sequence identity, or alternatively at least95% sequence identity, or alternatively at least 97% sequence identity,or alternatively at least 98% sequence identity to that shown in SEQ IDNO: 16 or one that hybridizes under stringent conditions to SEQ ID NO:16 or its complement. The choice of glycoprotein for use in the envelopeis determined in part, by the antibody to which the particle may beconjugated.

This invention also provides the suitable packaging cell line. In oneaspect, the packaging cell line is the HEK-293 cell line. Other suitablecell lines are known in the art, for example, described in the patentliterature within U.S. Pat. Nos. 7,070,994; 6,995,919; 6,475,786;6,372,502; 6,365,150 and 5,591,624, each incorporated herein byreference.

Pseudotyped Viral Particles

This invention further provides a method for producing a pseudotypedviral particle, comprising, or alternatively consisting essentially of,or yet further consisting of, transducing a packaging cell line with theviral system as described above, under conditions suitable to packagethe viral vector. Such conditions are known in the art and brieflydescribed herein. The pseudotyped viral particle can be isolated fromthe cell supernatant, using methods known to those of skill in the art,e.g., centrifugation. Such isolated particles are further provided bythis invention.

This invention further provides the isolated pseudotyped viral particleproduced by this method. The pseudotyped viral particle comprises, oralternatively consists essentially of, or yet further consists of apolynucleotide encoding a CCR5 RNAi and envelope protein comprising thepre-selected domain such as the ZZ S. aureus domain alone or incombination with a TRIM5alpha sequence and an HIV TAR decoypolynucleotide.

The isolated pseudotyped particles can be conjugated to one or more ofan antibody or an antibody fragment (e.g. an fragment containing atleast the Fc domain) that retains the ability to bind a pre-selectedcell receptor selected from the group consisting of CCR5, CD4, CD34 andCXCR4. Such antibodies include anti-CCR5 antibody, an anti-CD4 antibody,an anti-CD34 antibody and a CXCR4 antibody. Anti-CCR5 antibodies arecommercially available from Invitrogen; abcam; ProSci Inc. andeptitomics, and described in the patent literature within U.S. Pat. Nos.7,122,185; 7,175,988; 7,160,546; and 6,930,174, each incorporated hereinby reference. Anti-CD34 antibodies are commercially available from R&DSystems, Invitrogen; Biolegend; and Miltenyi Biotec, and described inthe patent literature within U.S. Pat. No. 4,965,204, incorporatedherein by reference. Anti-CD4 antibodies are commercially available fromR&D Systems, Invitrogen; Biolegend; and Miltenyi Biotec, and describedin the patent literature within U.S. Pat. No. 4,965,204, incorporatedherein by reference. Anti-CXCR4 antibodies are commercially availablefrom Leinco Technologies, Capralogics and BioLegend, and described inthe patent literature within U.S. Pat. Nos. 7,521,045; 6,949,243 and6,485,929, and U.S. Patent Publication No. 2005/0271665, eachincorporated herein by reference.

The antibodies are not species specific. In other words, the antibodiescan be polyclonal or monoclonal and can be murine, ovine, human or otherspecies. In addition, they can be chimeric or humanized.

When used in combination, the particles can be combination of CD4/CCR5or CD4/CXCR4 or CD4/CCR5/CXCR4 or CD4/CD34 or CD34/CCR5 or CD34/CXCR4 orCD4/CD34/CCR4/CXCR4, which target multiple populations of HIVsusceptible cells.

Methods to Produce the Pseudotyped Particles

This invention also provides methods to prepare a pseudotyped viralparticle by transducing a packaging cell line, as described herein withthe vector, the envelope plasmid and the packaging plasmid underconditions that facilitate packaging of the vector into the envelopeparticle. In one aspect, the pseudotyped viral particle is a pseudotypedviral particle. In a further aspect, the particles are separated fromthe cellular supernatant and conjugated to an antibody for cell-specifictargeting.

In one aspect, the complete vector particle is a viral, or alternativelya retroviral vector pseudotyped with a Sindbis virus glycoproteinenvelope containing the ZZ domain of Protein A from Staphylococcusaureus.

The genetic information of the viral vector particle is RNA whichcontains, on the 5′ and 3′ ends, the minimal LTR regions required forintegration of the vector. In between the two LTR regions is the psiregion which is required for packaging of the vector RNA into theparticle. This region is followed by the RRE and cPPT sequences whichenhance vector production by transporting the full length vectortranscript out of the nucleus for efficient packaging into the vectorparticle. Next is the polymerase-II promoter MNDU3 which drives theexpression of the chimeric TRIM5alpha gene. The polymerase-III U6promoter driven CCR5 shRNA gene follows immediately downstream. Next isthe polymerase-III U6 promoter driven TAR decoy gene. The last gene inthe vector is an EGFP gene (enhanced Green Fluorescent Protein) which isdriven by the polymerase-II PGK promoter. The EGFP gene is used as areporter gene to detect transduced cells. The above listed geneticelements are transcribed into a full length RNA molecule which ispackaged into the vector particle and contains all of the geneticinformation that will be integrated into the transduced cells.

The full length RNA transcript is packaged inside the capsid of thevector particle which contains the nucleocapsid, capsid, and matrixproteins which are generated from the packaging plasmid delta-8.91. Thereverse transcriptase polymerse which is generated from the packagingplasmid delta-8.91 is also located within the capsid with the RNAtranscript. The capsid encases and protects the full length RNAtranscript.

Surrounding the capsid/RNA complex is the Sindbis-ZZ glycoproteinenvelope which is generated from the Sindbis-ZZ plasmid. This envelope,when conjugated with a specific monoclonal antibody, will direct thevector particle to specifically transduce a cell of interest thatexpresses a cell surface receptor recognized by the chosen monoclonalantibody.

The vector particle is generated by a transient transfection protocolwhich includes a packaging cell line (HEK-293T cells), a lipofectionreagent (Transit-293T), and the three plasmids encoding the parts of thevector particle (delta-8.91 (packaging plasmid)), CD25-containingvectors described herein (viral vector plasmid), and Sindbis-ZZ(envelope plasmid).

HEK-293T cells are plated at 75% confluency in complete DMEM media 24hours prior to transfection. After at least 24 hours post-plating ofcells, the transfection mixture should be prepared. Three milliliters ofserum free media is incubated with 150 ul of the lipofection reagent for20 minutes at room temperature. The three plasmids are then added to themedia/lipofection reagent mixture at a ratio of 5:5:2 (packagingplasmid: viral vector plasmid: envelope plasmid) and incubated for 30minutes. After this final incubation period, the media/lipofectionreagent/DNA mixture is then added to the HEK-293T cells and leftovernight for the transfection to occur. The next day, the transfectionmedia is removed and fresh complete DMEM is added. Seventy-two hourslater, the cell culture supernatant is collected and concentrated byultracentrifugation at 20,000 rpm for 1.5 hours.

In one aspect, the 13-amino acid patch sequence required for theTRIM5alpha molecule to inhibit HIV infection is Nterminal—QAPGTLFTFPSLT—C terminal. Thus, any TRIM5alpha polynucleotidemust encode this primary amino acid sequence.

To construct the ZZ domain containing Sindbis virus glycoprotein, the ZZdomain from S. aureus was inserted in between the amino acids #71-#74 ofthe E2 region of the Sindbis glycoprotein gene. In the wild-type Sindbisglycoprotein E2 region, this is where normal cell surface receptorrecognition occurs. By inserting the ZZ domain here, normal cell bindingis abolished. After the ZZ domain was inserted into the E2 region in aBsteII restriction enzyme site, the entire E3-E2ZZ-6K-E1 glycoproteingene was PCR amplified and TOPO cloned into the pcDNA3.1 expressionplasmid. By inserting the glycoprotein genes into this expressionplasmid, the genes are under the control of the highly activepolymerase-II CMV promoter.

Once the vector particle buds from the packaging cells and is releasedinto the supernatant, this vector particle is conjugated to an antibodyas defined herein.

Isolated Host Cells

Yet further provided is an isolated cell or population of cells,comprising, or alternatively consisting essentially of, or yet furtherconsisting of, a retroviral particle of this invention, which in oneaspect, is a viral particle. In one aspect, the isolated host cell is apackaging cell line.

In another aspect, the invention provides a pseudotyped viral particleconjugated to an antibody as described herein which is furtherconjugated to a host cell expressing a receptor to which the viralparticle as described herein. In one aspect, the host cell is a cellexpressing one or more of CD4, CD34, CXCR4 and/or CCR5. The cell can beany of a cell of a species of the group of: murine, rats, rabbit,simians, bovines, ovine, porcine, canines, feline, farm animals, sportanimals, pets, equine, and primate, and in particular a human cell.Cells that are known to express such receptors include blood cell and inparticular lymphocyte cells such as peripheral blood lymphocytes andmobilized blood lymphocytes. In another aspect, the host cell is anadult stem cell such as an hematopoietic stem cell “HSC” (CD34⁺ and/orCD34⁺/Thy-1⁻ HSC).

In a further aspect is an isolated host cell expressing on its cellsurface CD25 extracellular domain or an equivalent thereof andcomprising, or alternatively consisting of, or yet further consisting ofCCR5 RNAi or an equivalent thereof. In a further aspect, the cellfurther comprises, or alternatively consists essentially of, or yetfurther consists of, a TRIM5alpha polynucleotic and/or HIR TARpolynucleotide or an equivalent of each thereof. The host cell canfurther comprise a detectable label. In one aspect the cell is a stemcell, such as a hematopoietic progenitor cell or a hematopoietic stemcell, e.g., a CD34+ cell. When used therapeutically, the cells can beallogeneic or autologous to the subject to be treated. The subjects canbe mammalian, e.g., murine, canine, bovine, equine, ovine, feline or ahuman subject or patient.

This invention also provides an enriched population of cells asdescribed above.

This invention further provides an isolated cell or an enrichedpopulation of cells that are derived from the stem cell described above.In one aspect, the derived cell or cell population is a macrophage.These cells are useful to treat and/or prevent HIV infection in asubject in need thereof.

Compositions, Screens and Therapeutic Uses

Also provided by this invention is a composition or kit comprising anyone or more of the compositions described above and a carrier, e.g., anisolated cell, an enriched population of cells, vectors, packagingsystem, pseudotyped viral, viral particle conjugated to an antibody orfragment thereof which in turn may optionally be conjugated to a celland a carrier. In one aspect, the carrier is a pharmaceuticallyacceptable carrier. These compositions can be used diagnostically ortherapeutically as described herein and can be used in combination withother known anti-HIV therapies.

The compositions can be used in vitro to screen for small molecules andother agents that may modify HIV infectivity and replication by addingto the composition varying amounts of the agent to be tested andcomparing it to a companion system that does not have the agent butwhich exhibits the desired therapeutic effect. For example, if it isknown that the viral particle or cell inhibits HIV infection in asystem, then the system can be used to test alternative therapies todetermine if it is a substitute to the viral particle or cell.Alternatively, one can test agents in the viral particle system itselfto determine if the agent acts competitively, additively orsynergistically with the viral particle system. After an in vitroscreen, the test agent or combination therapy can be assayed in anappropriate animal model.

When the cells, particles and/or antibody conjugated cells (to theparticles) are administered to an appropriate animal subject, the animalsubject can be used as an animal model to test alternative therapies inthe same manner as the in vitro screen. This invention also provides amethod to inhibit HIV replication in vivo or ex vivo, comprising, oralternatively consisting essentially of, or yet further consisting ofadministering to a subject in need thereof an effective amount of theisolated cell, enriched population of cells, pseudotyped viral particleor the pseudotyped viral particle conjugated to the antibody as describeherein. In another aspect, a method to prevent HIV replication in vivoor ex vivo is provided comprising, or alternatively consistingessentially of, or yet further consisting of administering to a subjectin need thereof an effective amount of the isolated cell, enrichedpopulation of cells, pseudotyped viral particle or the pseudotyped viralparticle conjugated to the antibody as describe herein. The cell,enriched population of cells, pseudotyped viral particle or thepseudotyped viral particle conjugated to the antibody as describe hereincan be combined with other anti-viral therapies that are known in theart. When combined with other therapies, administration of the therapiescan be concurrent or sequential as determined by the treating physician.In one aspect, bone marrow, mobilized bone marrow cells or peripheralblood lymphocytes are removed from the patient to be treated andcultured with the pseudotyped viral particle conjugated to the one ormore antibodies. After an appropriate amount of time to allow for theparticle to bind to the appropriate receptor, the cells are thenre-administered to the subject or patient to which they were isolated.As noted above, this therapy can be combined with other anti-viraltherapies or the like.

This invention also provides a method to treat a subject at risk ofdeveloping an active infection or infected with HIV (AIDs) byadministering to the subject an effective amount of one of thecompositions as described herein. For the purpose of this aspect, asubject is as described herein and therefore includes mammals, animalsand humans, for example. Additional effective therapies can combinedwith this invention and/or added as necessary.

Further provided are methods to inhibit or prevent HIV replication in acell infected with HIV, by contacting the cell with an effective amountof one or more of an isolated cell, an enriched population of cells, apseudotyped viral vector particle as described herein or or thepseudotyped viral vector as described herein. In one aspect, thecontacting is in vitro. In another aspect it is in vivo.

This invention also provides the use of a compositions as describedherein to prevent or treat an HIV infection and/or AIDs by administeringto a subject an effective amount of one or more compositions describedherein. Further provided is the use of a composition as described hereinin the manufacture of a medicament to treat or prevent HIV infectionand/or AIDs. Additional effective therapies can combined with thisinvention and/or added as necessary.

Having been generally described herein, the follow examples are providedto further illustrate this invention.

EXAMPLES CD25 Pre-Selective Anti-HIV Vectors for Improved HIV GeneTherapy

HIV infections continue to spread worldwide in both developed andunderdeveloped countries with no effective vaccine available (Barouch etal., 2008; Edgeworth et al., 2002). Although antiretroviral therapy(ART) is effective in the majority of HIV infected patients, challengesto therapeutic and curative success include the continuing emergence ofdrug-resistant HIV variants, drug toxicity, and incomplete viralsuppression (Baldanti et al., 2010; Domingo et al., 2012; Johnson etal., 2010; Kuritzkes, 2011; Lewden et al., 2007; Macias et al., 2006;Tilton et al., 2010). ART also fails to eradicate viral reservoirs whichare established early in infection, leading to viral persistence andincomplete immune restoration (Gazzola et al., 2009; Mehandru et al.,2006). Interruption of ART results in rapid viral resurgence, thegeneration of escape mutants, and CD4+ T cell loss in the peripheralblood of HIV infected patients (Graham et al., 2012; Kalmar et al.,2012).

These challenges highlight the need for the further development ofinnovative HIV therapies with broad mechanisms of action. HIV genetherapy has the potential as an alternative or complementary treatmentstrategy to ART, especially when hematopoietic stem cells (HSCs) are thecellular targets for genetic modification (Strayer et al., 2005).Advantages of HIV stem cell gene therapy include constitutive orcontrolled expression of anti-HIV genes, the generation of a durable andHIV-resistant immune system, and the possibility of a one-time treatmentif enough anti-HIV vector transduced cells can be transplanted intopatients (Strayer et al., 2005). Many potent anti-HIV genes have beendeveloped and tested both in preclinical and clinical settings and thesafety of some of these genes has been observed with engraftment oftransduced stem cells and anti-HIV gene expression in transplantedpatients (Bauer et al., 1997; DiGuisto et al., 2010; Kohn et al., 1999;Mitsuyasu et al., 2009; Podsakoff et al., 2005; Shimizu et al., 2010;ter Brake et al., 2009; Walker et al., 2012). However, efficacy in aclinical setting has been difficult to achieve due to low transductionefficiencies and low in vivo gene marking (DiGuisto et al., 2010;Mitsuyasu et al., 2009; Podsakoff et al., 2005).

In vitro HIV challenge experiments designed to evaluate the efficacy ofanti-HIV genes in inhibiting HIV infection/replication rely on sortingor selection of the gene transduced cells resulting in a pure populationof HIV-resistant cells prior to infection. However, this has not beenfeasible in a clinical setting as many reporter genes utilized forsorting may be immunoreactive. When unsorted/mixed populations ofnontransduced and anti-HIV vector transduced cells are infected withHIV, a selective survival advantage and an increase in the percentage oftotal immune cells of the anti-HIV gene expressing cells has beenobserved (Anderson et al., 2009; Walker et al., 2012). These resultshighlight the ability of anti-HIV gene modified cells to survive andpossibly replenish the immune system with functioning HIV-resistantimmune cells. However, when translated into a clinical setting, a largepercentage of nontransduced HSCs are transplanted along with theanti-HIV vector transduced cells due to low transduction efficiencies(DiGuisto et al., 2010; Mitsuyasu et al., 2009; Podsakoff et al., 2005).These nontransduced HSCs produce unprotected immune cells which aretargets for HIV infection and replication. These infected cells may alsoreplenish viral reservoirs. By transplanting an enriched population ofanti-HIV vector transduced cells into patients where the majority of thecells express the anti-HIV genes, similar results observed with theBerlin patient may be achievable as this patient received a purepopulation of HIV-resistant HSCs from a donor who was homozygous for aCCR5 Δ32 bp allele (Hutter et al., 2009). Therefore, new methods need tobe developed to increase the total percentage of anti-HIV vectortransduced cells transplanted into patients.

In the studies described herein, in vitro safety and an improvedefficacy of HIV stem cell gene therapy in the enriched population ofHIV-resistant cells compared to unpurified cells are demonstrated. Thiswas achieved by a triple combination anti-HIV vector which incorporateda selectable marker, human CD25, which is expressed on the surface oftransduced cells. Human CD25, the low affinity IL-2 receptor alphasubunit, was chosen as the selectable marker because of its normalcharacteristics of not being expressed on the surface of HPCs or HSCsand its lack of intracellular signaling (Grant et al., 1992; Kuziel etal., 1990; Minami et al., 1993). Upon expressing CD25 on the surface ofHPCs and purification of the transduced cells, safety of the enrichedpopulation of anti-HIV vector transduced HPCs was observed along withpotent HIV-1 inhibition. These results highlight the potential use ofthis strategy for HIV stem cell gene therapy to improve the efficacy offuture HIV stem cell gene therapy clinical trials.

Example 1: Lentiviral Vector Design and Production

A third-generation self-inactivating lentiviral vector was utilized inthis study, CCLc-MNDU3-X-PGK-X2. To generate the control vector (namedEGFP+), an EGFP reporter gene was inserted into position “X” under thecontrol of the MNDU3 promoter and a human CD25 coding region wasinserted into position “X2” of this vector under the control of aphosphoglycerate kinase (PGK) promoter (FIG. 1A). This vector was onlyused to initially test the strategy of utilizing CD25 as a selectiveprotein in purifying transduced cells. Therefore, Applicant was able tocompare EGFP % positive cells to CD25% positive cells. To generate thepre-selective anti-HIV vector (named CMAP1(Cclc-Mndu3-Antihiv-Protein-1), a triple combination of anti-HIV geneswas inserted into position “X” and a human CD25 coding region wasinserted into position “X2” of this vector under the control of a PGKpromoter (FIG. 1B). The triple combination of anti-HIV genes includes achimeric human/rhesus macaque TRIM5α gene under the control of the MNDU3promoter, a polymerase-III U6 promoter driven CCR5 shRNA expressioncassette, and a polymerase III U6 promoter driven TAR decoy expressioncassette (FIG. 1B). Sequencing of clones was confirmed by Laragen Inc.,Los Angeles, Calif.

Lentiviral vectors were generated in HEK-293T cells. Twenty-fivemicrograms of the packaging construct, Δ8.9 (packaging plasmidcontaining the gag and pol genes), 25 μg of EGFP+ or CMAP1, and 5 μg ofVSVG (envelope) were transfected into cells in T225 flasks bylipofection. Vector supernatants were collected at 48 hourspost-transfection and concentrated by ultrafiltration. Vector titerswere calculated by transduction of HEK-293T cells. Forty-eight hourspost-transduction, the HEK-293T cells were stained with a phycoerythrin(PE)-conjugated anti-human CD25 antibody (BD Biosciences, San Jose,Calif.) and analyzed by flow cytometry. All flow cytometry analyses wereperformed on a Beckman Coulter Cytomics FC500 using CXP software.

Example 2: Transduction and Purification of Vector Transduced PrimaryHuman CD34+ HPCs

CD34+ hematopoietic progenitor cells (HPCs) were isolated from humanumbilical cord blood (NDRI, Philadelphia, Pa.) by Ficoll-Paque (GEHealthcare, Piscataway, N.J.) and purified by CD34+ magnetic bead columnseparation (Miltenyi Biotec, Auburn, Calif.). CD34+ cell isolationpurity (>90%) was routinely obtained. Total CD34+ cells were cultured incomplete IMDM media containing 10% FBS and supplemented with 50 ng/mlSCF, Flt-3 ligand, and TPO. Cells were transduced with the lentiviralvectors EGFP+ or CMAP1 (MOI 15) overnight at 37° C. with 8 μg/mlprotamine sulfate. Two days post-transduction, an aliquot of the EGFP+vector transduced cells was stained with a PE-conjugated anti-human CD25antibody (BD Biosciences, San Jose, Calif.) and analyzed by flowcytometry for both EGFP and CD25 percentages. Total genomic DNA wasextracted from an aliquot of the CMAP1 vector transduced cells utilizinga Wizard Genomic DNA Isolation System (Promega, Madison, Wis.) andanalyzed by quantitative PCR (QPCR) for vector copy number with a primerset specific for the chimeric TRIM5α gene: (forward)5′-CTGGGTTGATGTGACAGTGG-3′ (SEQ ID NO: 28) and (reverse)5′-CGTGAGTGACGGAAACGTAA-3′ (SEQ ID NO: 29). QPCR was performed using aSYBR Green PCR Master Mix Kit (Applied Biosystems, Foster City, Calif.).The rest of the transduced cells were labeled with CD25+ immunomagneticbeads (Miltenyi Biotec, Auburn, Calif.) according to the manufacturer'sprotocol and separated over a magnetic bead column. Purified cells werethen utilized for subsequent experiments.

To evaluate the purity of the EGFP+vector transduced cells after CD25+immunomagnetic bead selection, purified cells were stained with aPE-conjugated anti-human CD25 antibody (BD Biosciences, San Jose,Calif.) and analyzed for both EGFP and CD25 percentages. To evaluate thepurity of the CMAP1 vector transduced cells, total genomic DNA wasextracted and analyzed by QPCR for vector copy number utilizing thechimeric TRIM5α primer set described above. QPCR was performed using aSYBR Green PCR Master Mix Kit (Applied Biosystems, Foster City, Calif.).GAPDH was used as an internal control.

Example 3: Colony Forming Unit Assays

CD34+ HPCs, either nontransduced (NT) or CD25 immunomagnetic beadpurified CMAP1 vector transduced cells were cultured in semi-solidmethylcellulose medium with growth factors (Stem Cell Technologies,Vancouver, BC, Canada) for 10 days. After 10 days, total blood formingcolonies (BFU), granulocyte/erythrocyte/megakaryocyte/monocyte colonies(GEMM), and granulocyte/monocyte colonies (GM) were observed andcounted.

To evaluate whether purified CMAP1 vector transduced CD34+ HPCs (10,000cells/3 ml methylcellulose) had an increased proliferation potential inthe presence of IL-2, 1 μg/ml of IL-2 was added to the methylcellulosemedium. After 10 days, total cell numbers were counted.

To derive macrophages from the nontransduced and the purified CMAP1vector transduced CD34+ HPCs, cells were removed from themethylcellulose and plated in 6-well plates in complete DMEM medium with10% FBS supplemented with 10 ng/ml of GM-CSF and M-CSF (R&D Systems,Minneapolis, Minn.). Media was changed every two days for four days toderive mature macrophages. Both nontransduced (NT) and purified CMAP1vector transduced CD34+ cell derived macrophages were used forsubsequent experiments.

Example 4: Phenotypic and Genotypic Analysis of CD34+ Cell DerivedMacrophages

To determine if CMAP1 vector transduced CD34+ cells were able to matureinto phenotypically normal macrophages, cells were visualized bymicroscopy and analyzed by flow cytometry. Macrophages were stained withantibodies to detect normal macrophage cell surface markers including,PE-conjugated CD14, allophycocyanin (APC)-conjugated HLADR,PE-conjugated CD4, PECY7-conjugated CD80, PE-conjugated CCR5, andPE-conjugated CD25 (BD Biosciences, San Jose, Calif.).

To determine if the addition of IL-2 induced the expression ofproto-oncogenes in CMAP1 vector transduced macrophages, IL-2 (1 μg/ml)was added to the macrophage cultures. On day three post-IL2 addition,total cellular RNA was extracted and QPCR was performed. Total cellularRNA was extracted from cells using RNA-STAT-60 (Tel-Test Inc.,Friendswood, Tex.). First strand cDNA synthesis was performed using theHigh Capacity cDNA Reverse Transcription Kit (Applied Biosystems, FosterCity, Calif.). QPCR was then performed using the SYBR Green PCR MasterMix Kit (Applied Biosystems, Foster City, Calif.) with primers: myc(forward) 5′-TCCATTCCGAGGCCACAGCAAG-3′ (SEQ ID NO: 30); (reverse)5′-TCAGCTCGTTCCTCCTCTGACG-3′ (SEQ ID NO: 31); myb (forward)5′-AAGACCCTGAGAAGGAAAAGCG-3′ (SEQ ID NO: 32); (reverse)5′-GTGTTGGTAATGCCTGCTGTCC-3′ (SEQ ID NO: 33); fos (forward)5′-ACTACCACTCACCCGCAGAC-3′ (SEQ ID NO: 34); (reverse)5′-GACGGGAAGCCAGCCTTAC-3′ (SEQ ID NO: 35); jun (forward)5′-CCCCAAGATCCTGAAACAGA-3′ (SEQ ID NO: 36); and (reverse)5′-CCGTTGCTGGACTGGATTAT-3′ (SEQ ID NO: 37). Glyceraldehyde-6-phosphatewas used as an internal control. Peripheral blood mononuclear cells(PBMCs) were cultured in complete RPMI media supplemented with 10% FBSand used as a positive control for proto-oncogene up-regulation uponIL-2 (1 μg/ml) stimulation.

Example 5: HIV-1 Challenge of Vector Transduced Macrophages

To determine whether the purified CMAP1 vector transduced macrophageswere capable of improved inhibition of HIV-1 infection compared tounpurified CMAP1 vector transduced macrophages, cells were challengedwith an R5-tropic BaL-1 strain of HIV-1 (MOI 0.05). On various dayspost-infection, supernatants were collected and analyzed for levels ofHIV-1 replication by p24 antigen ELISA (Zeptometrix Corp., Buffalo,N.Y.). On day 28 post-infection, the HIV-1 challenged cells were alsovisualized by microscopy.

Two-sample t-tests were used for statistical analyses. They wereconducted in R (version 2.10.1) for Windows. A significance level of0.05 was used for testing hypotheses.

Example 6: Enrichment of Cells Transduced with the Pre-Selective Vectors

A third generation self-inactivating lentiviral vector,CCLc-MNDU3-X-PGK-X2, was utilized to derive the control (EGFP+) and theanti-HIV (CMAP1) pre-selective vectors. A selectable marker, human CD25which is not normally found on the surface of CD34+ HPCs, wasincorporated into the control EGFP+ and CMAP1 vectors under the controlof the PGK promoter and used to purify vector transduced cells (FIG.1A). The EGFP+ control vector was only used to initially test thestrategy of utilizing CD25 as a selective protein in purifyingtransduced cells and to compare the purification levels to CMAP1 vectortransduced cells. By using EGFP in the same vector as CD25, we would beable to compare EGFP % positive cells to CD25% positive cells. Toevaluate the levels of purification of vector transduced cells, flowcytometry and quantitative PCR (QPCR) were performed on EGFP+ and CMAP1vector transduced CD34+ HPCs, respectively. As displayed in FIG. 1B, thepercentage of control EGFP+ vector transduced cells in the total cellpopulation increased on average from 14.5% EGFP+ unpurified to 81.9%EGFP+ after purification. This correlated with expression of CD25 on thesurface of the transduced HPCs which increased on average from 15.1%CD25+ before purification to 81.0% CD25+ post-purification. As displayedin FIG. 1C, the levels of vector copy number per cell in the total cellpopulation of CMAP1 vector transduced cells increased on average from0.38 vector copies per cell unpurified to 2.97 vector copies per cellafter purification. This amounted to an average enrichment percentage of85.3%. These data successfully demonstrate that upon expression of CD25on the surface of cells transduced with the pre-selective vectors, ahigh level of enrichment can be achieved.

Example 7: Safety of CMAP1 Transduced HPCs in CFU Assays

CD25 is not normally found on the surface of human HPCs. Therefore, toevaluate the effect of CD25 expression on HPCs and their ability to formnormal quantities and types of hematopoietic colonies, CFU assays wereperformed on purified CMAP1 vector transduced HPCs. As displayed in FIG.2A, normal colony phenotypes of BFUs, GEMMs, and GMs formed in thepurified CMAP1 cultures compared to nontransduced (NT) cultures. Therepresentative pictures displayed were taken with a 10× objective. Totalnumbers of each type of colony were also counted. As displayed in FIG.2B, no significant differences (p>0.05) in the numbers of each colony,BFUs, GMs, and GEMMs had formed, on average, in the purified CMAP1cultures, 44.0 BFUs, 45.3 GMs, and 12.0 GEMMs with standard deviationsof 5.29, 5.03 and 2.00, respectively compared to the NT cultures, 45.3BFUs, 50.0 GMs, and 12.7 GEMMs with standard deviations of 3.06, 5.29and 5.03, respectively.

As CD25 is part of the IL-2 receptor complex which is involved in theimmune response and immune cell proliferation, we wanted to investigatewhether the addition of IL-2 to the purified CMAP1 vector transduced HPCmethylcellulose cultures resulted in an increase in HPC proliferationand in total cell numbers. As displayed in FIG. 2C, no significantdifferences (p>0.05) in cell numbers were observed, on average, in theNT and CMAP1 cultures with the addition of IL-2 (106.74 and 106.78 totalcells, respectively). An increase in total cell numbers was observed inthe CMAP1 cultures with IL-2 compared to the cultures without IL-2 (anincrease of 0.18 log), however, a similar increase was observed in theNT cultures (an increase of 0.16 log). This was likely due to thepresence of cells which normally respond to IL-2 since during the courseof the 10 day culture, the majority of the cells have differentiated.

These results highlight the initial safety of expressing CD25 on thesurface of vector transduced HPCs and demonstrated that no apparentaberrations in HPC differentiation or proliferation had occurred withover expression of CD25.

Example 8: Derivation of Phenotypically Normal Macrophages from CMAP1Transduced HPCs

To determine whether normal immune cells could be derived from thepurified CMAP1 transduced cells, macrophages were differentiated fromthe HPCs. Macrophage cultures were visualized by microscopy under a 10×objective. As displayed in FIG. 3A, normal macrophage phenotypes wereobserved with the appearance of attached cells having a “fried-egg”appearance in both the NT and purified CMAP1 vector transduced cultures.These macrophages were then phenotypically analyzed by flow cytometryfor normal macrophage cell surface markers. As displayed in FIG. 3B, anormal phenotype of purified CMAP1 macrophages (shaded histograms) wasdemonstrated as compared to NT macrophages (unshaded histograms).Representative overlay histograms are displayed (FIG. 3B). CMAP1 cellsdisplayed 92.3% of CD14, 95.6% of HLADR, 99.7% of CD4, and 93.4% ofCD80. CCR5 expression had decreased to 25.5% in the purified CMAP1macrophages compared to NT macrophages which displayed a level of 94.6%of CCR5. The decrease in CCR5 expression in the CMAP1 cultures was dueto the expression of the CCR5 shRNA.

Macrophages normally express a certain level of CD25 on their cellsurface. To evaluate the increase in cell surface expression of CD25 onCMAP1 transduced macrophages, flow cytometry was performed. As displayedin FIG. 3C, a gradient increase was observed, on average, in CD25expression on the surface of macrophages from NT macrophages (55.0%) tounpurified CMAP1 transduced macrophages (79.1%) to purified CMAP1transduced macrophages (95.1%).

These results demonstrate that phenotypically normal macrophages can bederived from purified CMAP1 vector transduced HPCs and thatoverexpression of CD25 did not disrupt normal macrophage differentiationfrom HPCs. These results also demonstrate that the CMAP1 vector workedas hypothesized by displaying an increase in CD25 expression on purifiedpopulations of transduced cells.

Example 9: Proto-Oncogene Expression in Purified CMAP1 Macrophages

As mentioned above, CD25 is part of the IL-2 receptor complex which isinvolved in the cell proliferation of immune cells. After IL-2 binds tothe IL-2 receptor, expression of proto-oncogenes including myc, myb,fos, and jun are up-regulated to promote cell division. To evaluatewhether overexpression of CD25 on the surface of purified CMAP1macrophages up-regulated the expression of the mentionedproto-oncogenes, QPCR was performed on total RNA from macrophagecultures with the addition of IL-2. PBMCs were used as a positivecontrol for proto-oncogene up-regulation in the presence of IL-2. Asdisplayed in FIG. 4, similar levels of proto-oncogene expression weremeasured in NT and purified CMAP1 macrophage cultures. No significantdifference (p>0.05) was observed with the expression of myc, myb, andfos in CMAP1 macrophages compared to NT macrophages in the presence ofIL-2. However, an average relative expression level of 1.1 (standarddeviation of 0.04) for jun (p=0.0121, statistically significant) wasobserved with CMAP1 macrophages compared to NT macrophages which wereused as the reference cells with an average relative expression level of1.0 (standard deviation of 0.04). As a positive control forproto-oncogene up-regulation, PBMCs were cultured in the presence ofIL-2. A significant up-regulation in the expression of myc (2.3-fold)(p=0.0418), myb (8.1-fold) (p=0.0018), fos (4.8-fold) (p=0.0001), andjun (5.3-fold) (p=0.0004) was observed in the IL-2 stimulated PBMCcultures relative to NT macrophages.

These results demonstrate that even though the purified CMAP1macrophages express an increased level of CD25, proto-oncogeneexpression levels remained similar to nontransduced cells.

Example 10: HIV-1 Inhibition of CMAP1 HPC Derived Macrophages

Previous challenge experiments performed with this triple combination ofanti-HIV genes, both in vitro and in vivo, demonstrated strong viralinhibition to both CCR5 and CXCR4-tropic strains of HIV-1 (Anderson etal., 2009; Kohn et al., 1999). These experiments, however, relied onsorting the cells based on a non-clinically acceptable reporter gene,EGFP, prior to viral challenge. To evaluate whether purified CMAP1macrophages displayed an increased efficacy of HIV-1 inhibition comparedto NT and unpurified CMAP1 macrophages, cells were challenged with anR5-tropic BaL-1 strain of HIV-1. As displayed in FIG. 5A at the end ofthe challenge experiments, potent inhibition of HIV-1 infection wasobserved in the purified CMAP1 cultures compared to NT macrophages (2.9log difference) and unpurified CMAP1 macrophages (2.2 log difference). Aslight inhibition of HIV-1 infection was observed in the unpurifiedCMAP1 macrophage cultures (0.7 log difference) compared to NTmacrophages due to the presence of anti-HIV gene expressing cells. Onday 28-post infection, HIV-1 infected macrophages were also visualizedby microscopy under 10× magnification. As displayed in FIG. 5B withrepresentative pictures, cell death from HIV infection was observedthroughout the cultures of infected NT and unpurified CMAP1 macrophages.This was in comparison to purified CMAP1 macrophages which displayed asmall amount of cell death but where the majority of macrophages stillappeared healthy.

These results highlight the increased efficacy of anti-HIV vectortransduced cells when they are purified to a cell population where themajority of the cells express the anti-HIV genes and demonstrate theutility of this novel CMAP1 vector.

HIV gene therapy holds considerable promise as an alternative treatmentstrategy for HIV infected patients. As observed with the Berlin patientwho received a pure population of HIV-resistant hematopoietic stem cellsin a bone marrow transplant from a donor homozygous for a CCR5 Δ32 bpallele, HIV-resistant stem cells are capable of repopulating the immunesystem with cells which can inhibit HIV infection in the absence of ARTfor a prolonged period of time (Hutter et al., 2009). The safety ofnumerous anti-HIV genes has been demonstrated in previous HIV stem cellgene therapy clinical trials (DiGuisto et al., 2010; Mitsuyasu et al.,2009; Podsakoff et al., 2005). However, patients were given a mixedpopulation of cells, with the vast majority not being transduced withanti-HIV genes. This led to the derivation of an immune system where themajority of the immune cells were still susceptible to HIV infection. Alow level of efficacy was observed with a measureable increase in thelevels of anti-HIV gene expressing cells in the presence of a viralload, however, initial transduction efficiencies and in vivo genemarking were too low for patients to remain off ART. If a purepopulation of anti-HIV gene transduced cells could be transplanted intopatients, similar results observed with the Berlin patient may beachievable.

Applicant has recently demonstrated strong ex vivo resistance toinfection to multiple strains of HIV-1 and a selective survivaladvantage of cells transduced with a triple combination anti-HIVlentiviral vector expressing a human/rhesus macaque TRIM5α, a CCR5shRNA, and a TAR decoy in vivo in a humanized mouse model (Walker etal., 2012). However, plasma viremia levels in HIV-1 infected micetransplanted with anti-HIV vector transduced cells did not decrease overtime due to the transplantation of a majority of nontransduced HSCswhich continually produced HIV susceptible target cells (Walker et al.,2012). If anti-HIV vector transduced cells could be enriched to a purepopulation or at least to a population where the majority of the cellsexpress the anti-HIV genes, improved in vivo efficacy may bedemonstrated.

Current reporter genes for cell sorting are not clinically relevant dueto their foreign nature which would invoke an immune response againsttransduced cells. Another approach similar to the one presented hereutilizes a P140K mutant methylguanine methyltransferase (MGMT) transgeneto select for transduced cells in vivo after transplantation intopatients. This, however, would require another patient infusion withagents which would select for vector transduced cells (Trobridge et al.,2009). Therefore, as a first step to improve on the efficacy of HIV genetherapy, Applicant has developed pre-selective anti-HIV lentiviralvectors which express a normal human cell surface protein as to avoidrejection of transduced cells and allow for the purification of vectortransduced cells prior to transplantation. Human CD25 was chosen as aselectable marker based on its characteristics of not being expressed onthe surface of HPCs or HSCs, it is a normal immune cell surface protein,and it has been previously shown to have no direct intracellularsignaling (Grant et al., 1992; Kuziel et al., 1990; Minami et al.,1993).

When over-expressing a protein on the surface of cells, especially cellswhich do not normally express the protein, the safety and function ofthe engineered cells is a concern. Upon transduction and high levelpurification (>85%) of CMAP1 vector transduced HPCs, phenotypicallynormal CFUs and the number of CFUs formed in methylcellulose medium weresimilar to nontransduced cells (FIG. 2). Safety was also observed withmacrophages derived from the purified CMAP1 vector transduced HPCs asthey appeared phenotypically normal compared to nontransducedmacrophages (FIG. 3).

As CD25 is part of the IL-2 receptor complex, it was important toinvestigate whether the over expression of CD25 in purified vectortransduced cells had any effect on cell proliferation or theup-regulation of proto-oncogenes in the addition of IL-2. In thepresence of IL-2 no adverse effects were observed in purified CMAP1vector transduced HPCs or macrophages. No increased cell proliferationof HPCs (FIG. 2) and no increase in the expression of theproto-oncogenes myc, myb, or fos (FIG. 4) was observed compared tonontransduced cells. A small increase in the expression of jun in CMAP1vector transduced macrophages was observed (1.1) compared to NT cells(1.0). This difference was, however, significantly different to thepositive control PBMCs which displayed a 5.3 relative increase inexpression of jun. No other adverse effects were observed with purifiedCMAP1 vector transduced cells. Applicant's findings of a lack of cellproliferation and proto-oncogene up-regulation are likely due to CD25'snormal function of being involved with the assembly of the IL-2 receptorbut having no mitotic signaling capabilities (Grant et al., 1992; Kuzielet al., 1990; Minami et al., 1993).

The ultimate goal of HIV stem cell gene therapy is to provide analternative therapeutic intervention for HIV infected patients and toprovide a possibility for them to withdraw ART medications. For this tobe achievable, an enriched population of anti-HIV gene transduced cellswith little to no nontransduced cells needs to be transplanted. UponHIV-1 challenge of purified CMAP1 vector transduced HPC derivedmacrophages, strong inhibition of HIV-1 infection was observed. CMAP1vector transduced cells which were not purified displayed high levels ofHIV-1 replication similar to nontransduced macrophage cultures. Eventhough unpurified CMAP1 cultures contained anti-HIV gene expressingcells, the majority of the cells were nontransduced and, thus, werecapable of being infected and replicating HIV-1 at a high level. Thishighlights the improved efficacy of this pre-selective anti-HIV vectorand allows for the purification of anti-HIV gene expressing cells to anenriched population.

Example 11: CD25-Pre-Selective-Anti-HIV Lentiviral Vector

Construction of the lentiviral vector depicted in FIG. 9 was performedaccording to the following. The lentiviral vector backbone CCLc was usedto generate the CD25 pre-selective anti-HIV vectors. First, ahuman/rhesus macaque TRIM5alpha gene was fused via a 2A proteasecleavage site to either the complete human CD25 gene or to a truncatedversion of the human CD25 gene (either a full truncation of thecytoplasmic domain or a truncation which leaves only four cytoplasmicamino acids). These fusion proteins were generated by fusion PCR andcontained an HpaI site at the 3′ end of the fusion gene for subcloningof the U6-CCR5 shRNA/U6-TAR decoy expression cassettes. TheTRIM5alpha-2A-CD25 fusion genes were first cloned into a TOPO PCRcloning vector and the sequence was verified. Next, the U6-CCR5shRNA/U6-TAR decoy expression cassettes were subcloned into an HpaI sitedownstream from the TRIM5alpha-2A-CD25 genes in their respective TOPOclones. Correct sequences were verified. Next, theTRIM5alpha-2A-CD25-U6-CCR5 shRNA/U6 TAR decoy expression clusters weresubcloned into the CCLc lentiviral vector in an EcoRI site downstream ofthe MNDU3 promoter. Sequences were verified. This generated the CCLclentiviral vector which contains the TRIM5alpha-2A-CD25 expressioncassette under the control of the MNDU3 promoter, a CCR5 shRNA under thecontrol of a U6 promoter, and a TAR decoy under the control of a U6promoter. SEQ ID NOS 1-3 are representative sequences of this vectorconstruct.

Example 12: Derivation of Macrophages from Purified CMAP1 VectorTransduced HPCs

Macrophages normally express a measureable level of CD25 on their cellsurface. To evaluate any increase in cell surface expression of CD25 onCMAP1-transduced macrophages, flow cytomotry was performed. A gradientincrease was observed, on average, in CD25 expression on the surface ofmacrophages from NT macrophages (63.7%) to unpurified CMAP1-transducedmacrophages (84.1%) to purified CMAP1-transduced macrophages (94.3%).This increase in CD25 expression, which correlates to CMAP1 vectortransduction, resulted in the down regulation of CCR5 due to theexpression of the CCR5 shRNA. In purified CMAP1 macrophages, CCR5expression was 12.5%, which compared to NT macrophages (86.5%) andunpurified CMAP1 macrophages (78.9%).

These results demonstrate that phenotypically normal macrophages can bederived from purified CMAP1 vector-transduced HPCs and thatoverexpression of CD25 did not disrupt normal macrophage differentiationfrom HPCs.

It is to be understood that while the invention has been described inconjunction with the above embodiments, that the foregoing descriptionand examples are intended to illustrate and not limit the scope of theinvention. Other aspects, advantages and modifications within the scopeof the invention will be apparent to those skilled in the art to whichthe invention pertains.

In addition, where features or aspects of the invention are described interms of Markush groups, those skilled in the art will recognize thatthe invention is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

All publications, patent applications, patents, and other referencesmentioned herein are expressly incorporated by reference in theirentirety, to the same extent as if each were incorporated by referenceindividually. In case of conflict, the present specification, includingdefinitions, will control.

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1. A vector comprising: (a) a backbone comprising essential sequencesfor integration into a target cell genome; (b) a nucleic acid encoding aCCR5 RNAi; (c) a first expression control element that regulatesexpression of the nucleic acid encoding the RNAi of element (b); (d) anucleic acid encoding at least the extracellular domain of CD25; and (e)a second expression control element that regulates expression of thenucleic acid encoding at least the extracellular domain of CD25.
 2. Thevector of claim 1, wherein the first or second expression controlelement is a promoter.
 3. (canceled)
 4. The vector of claim 1, furthercomprising a nucleic acid encoding TRIM5alpha sequence under the controlof (i) a third control element or (ii) the second expression controlelement.
 5. (canceled)
 6. The vector of claim 1, further comprising anucleic acid encoding an HIV TAR sequence and a promoter that regulatesexpression of the nucleic acid.
 7. (canceled)
 8. (canceled)
 9. Thevector of claim 1, wherein the CCR5 RNAi is one or more of the groupshRNA, siRNA, miRNA or dsRNA. 10-15. (canceled)
 16. A viral packagingsystem comprising: (a) the vector of claim 1, wherein the backbone isderived from a virus; (b) a packaging plasmid; and (c) an envelopeplasmid.
 17. The viral packaging system of claim 16, wherein theenvelope plasmid comprises a VSVG envelope protein or a functionalportion thereof. 18-20. (canceled)
 21. A method for producing apseudotyped viral particle, comprising transducing a packaging cell linewith the system of claim 16 under conditions suitable to package theviral vector.
 22. (canceled)
 23. A pseudotyped viral particle producedby the method of claim
 21. 24. The pseudotyped viral particle of claim16, further comprising a viral vector comprising a polynucleotideencoding a CCR5 RNAi and an envelope protein comprising a VSVG envelopeprotein or a functional portion thereof. 25-26. (canceled)
 27. A methodto inhibit HIV replication in a cell infected with HIV, comprisingcontacting the cell with an effective amount of the pseudotyped viralvector particle of claim
 23. 28. A method to inhibit HIV replication,comprising administering to a subject in need thereof an effectiveamount of the pseudotyped viral vector particle of claim
 23. 29. Avector comprising: (a) a backbone comprising essential nucleic acidsequences for integration into a target cell genome; (b) a nucleic acidencoding a CCR5 RNAi; (c) a nucleic acid encoding a TRIM5alpha sequence;(d) a nucleic acid encoding an HIV TAR sequence; (e) a nucleic acidencoding at least the extracellular domain of CD25; and (f) one or moreexpression control elements that regulate expression of the nucleic acidencoding the elements (b), (c), (d) and (e).
 30. The vector of claim 29,wherein at least one of the expression control elements is a promoter.31. The vector of claim 30, wherein the one or more expression controlelement comprises a Polymerase III promoter or a Polymerase II promoterthat regulates expression of the CCR5 RNAi or the HIV TAR sequence.32-56. (canceled)
 57. A viral packaging system comprising: (a) thevector of claim 29, wherein the backbone is derived from a virus; (b) apackaging plasmid; and (c) an envelope plasmid.
 58. The viral packagingsystem of claim 57, wherein the envelope plasmid comprises a VSVGenvelope protein or a functional portion thereof. 59-61. (canceled) 62.A method for producing a pseudotyped viral particle, comprisingtransducing a packaging cell line with the system of claim 57 underconditions suitable to package the viral vector.
 63. The method of claim62, further comprising isolating the pseudotyped viral particle. 64.(canceled)
 65. A pseudotyped viral particle produced by the method ofclaim
 62. 66. A pseudotyped viral particle comprising a viral vectorcomprising: a polynucleotide encoding a CCR5 RNAi, a polynucleotideencoding a TRIM5alpha sequence or an equivalent thereof, apolynucleotide encoding an HIV TAR sequence or an equivalent thereof, apolynucleotide encoding at least the extracellular domain of CD25 or anequivalent thereof, and envelope protein comprising a VSVG envelopeprotein or a functional portion thereof.
 67. The pseudotyped viralparticle of claim 23, further comprising one or more antibodiesconjugated to the envelope that may be the same or different. 68-70.(canceled)
 71. A method to inhibit HIV replication in a cell infectedwith HIV, comprising contacting the cell with an effective amount of thecell of the pseudotyped viral vector particle of claim
 65. 72. A methodto inhibit HIV replication, comprising administering to a subject inneed thereof an effective amount of the pseudotyped viral vectorparticle of claim
 65. 73. A method to prevent HIV replication,comprising administering to a subject in need thereof an effectiveamount of the pseudotyped viral vector particle of claim
 65. 74. Themethod of claim 71, wherein the contacting is in vitro or in vivo.
 75. Amethod to treat an HIV infection in a subject in need thereof,comprising administering to the subject an effective amount of a cellconjugated to the pseudotyped viral particle of claim
 66. 76-77.(canceled)
 78. A kit for use in treating an HIV infection in a subjectin need thereof, comprising the pseudotyped viral vector particle ofclaim 62, and instructions to use.
 79. A method to treat an HIVinfection in a subject in need thereof, comprising administering to thesubject an effective amount of a cell conjugated to the pseudotypedviral particle of claim
 21. 80-81. (canceled)
 82. A kit for use intreating an HIV infection in a subject in need thereof, comprising thepseudotyped lentiviral vector particle of claim 21, and instructions touse.